Pd-l2 variant immunomodulatory proteins and uses thereof

ABSTRACT

Provided herein are immunomodulatory proteins comprising variant PD-L2 and nucleic acids encoding such proteins. The immunomodulatory proteins provide therapeutic utility for a variety of immunological and oncological conditions. Compositions and methods for making and using such proteins are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. provisional application No. 62/472,572 filed Mar. 16, 2017, entitled “PD-L2 Variant Immunomodulatory Proteins and Uses Thereof,” U.S. provisional application No. 62/475,156 filed Mar. 22, 2017, entitled “PD-L2 Variant Immunomodulatory Proteins and Uses Thereof,” and U.S. provisional application No. 62/537,928 filed Jul. 27, 2017, entitled “PD-L2 Variant Immunomodulatory Proteins and Uses Thereof,” the contents of each of which are incorporated by reference in their entirety.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 761612001540SeqList.TXT, created Mar. 12, 2018 which is 3,353,517 bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.

FIELD

The present disclosure relates to therapeutic compositions for modulating immune response in the treatment of cancer and immunological diseases. In some aspects, the present disclosure relates to particular variants of PD-L2 that exhibit improved binding, such as improved binding affinity or selectivity, for one or more of the cognate binding partner proteins PD-1 and RGMb.

BACKGROUND

Modulation of the immune response by intervening in the processes that occur in the immunological synapse (IS) formed by and between antigen-presenting cells (APCs) or target cells and lymphocytes is of increasing medical interest. Mechanistically, cell surface proteins in the IS can involve the coordinated and often simultaneous interaction of multiple protein targets with a single protein to which they bind. IS interactions occur in close association with the junction of two cells, and a single protein in this structure can interact with both a protein on the same cell (cis) as well as a protein on the associated cell (trans), likely at the same time. Although therapeutics are known that can modulate the IS, improved therapeutics are needed. Provided are immunomodulatory proteins, including soluble proteins or transmembrane immunomodulatory proteins capable of being expressed on cells, that meet such needs.

SUMMARY

Provided herein is a variant PD-L2 polypeptide, containing an IgV domain or a specific binding fragment thereof, an IgC domain or a specific binding fragment thereof, or both, wherein the variant PD-L2 polypeptide contains one or more amino acid modifications in an unmodified PD-L2 or a specific binding fragment thereof corresponding to position(s) selected from 2, 12, 13, 15, 18, 20, 23, 24, 28, 31, 32, 33, 36, 37, 39, 44, 45, 46, 47, 48, 58, 59, 65, 67, 69, 71, 72, 73, 74, 75, 76, 77, 82, 85, 86, 89, or 91 with reference to numbering of SEQ ID NO:31. In some embodiments, the amino acid modification is an amino acid substitution, insertion or deletion.

In some embodiments, the unmodified PD-L2 is a mammalian PD-L2 or a specific binding fragment thereof. In some of any such embodiments, the unmodified PD-L2 is a human PD-L2 or a specific binding fragment thereof. In some of any such embodiments, the unmodified PD-L2 contains (i) the sequence of amino acids set forth in SEQ ID NO:31, (ii) a sequence of amino acids that has at least 95% sequence identity to SEQ ID NO:31; or (iii) a portion thereof containing an IgV domain or IgC domain or specific binding fragments thereof or both.

In some of any such embodiments, the specific binding fragment of the IgV domain or IgC domain has a length of at least 50, 60, 70, 80, 90, 100, 110 or more amino acids; or the specific binding fragment of the IgV domain contains a length that is at least 80% of the length of the IgV domain set forth as amino acids 24-130 of SEQ ID NO:4 and/or the specific binding fragment of the IgC domain contains a length that is at least 80% of the length of the IgC domain set forth as amino acids 122-203 of SEQ ID NO:4. In some of any such embodiments, the variant PD-L2 contains up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid modifications, optionally amino acid substitutions, insertions and/or deletions. In some of any such embodiments, the variant PD-L2 polypeptide contains a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:31 or a specific binding fragment thereof.

In some of any such embodiments, the variant PD-L2 polypeptide exhibits altered binding to the ectodomain of PD-1 or RGMb compared to the binding of the unmodified PD-L2. In some of any such embodiments, the variant PD-L2 polypeptide exhibits altered binding to the ectodomain of PD-1 compared to the unmodified PD-L2. In some aspects, the altered binding is altered binding affinity and/or altered binding selectivity.

In some of any such embodiments, the one or more amino acid modifications are selected from F2L, I12V, I13V, H15Q, N18D, N24S, C23S, G28V, N24D, V31A, V31M, N32D, L33P, L33H, L33F, I36V, T37A, S48C, S39I, E44D, N45S, D46E, T47A, E58G, E59G, K65R, S67L, H69L, P71S, Q72H, V73A, Q74R, V75G, R76G, D77N, Q82R, I85F, I86T, V89D, W91R, or a conservative amino acid substitution thereof. In some of any such embodiments, the one or more amino acid modifications are selected from among H15Q, N24D, E44D, V89D, Q82R/V89D, E59G/Q82R, S39I/V89D, S67L/V89D, S67L/I85F, S67L/I86T, H15Q/K65R, H15Q/Q72H/V89D, H15Q/S67L/R76G, H15Q/R76G/I85F, H15Q/T47A/Q82R, H15Q/Q82R/V89D, H15Q/C23S/I86T, H15Q/S39I/I86T, E44D/V89D/W91R, I13V/S67L/V89D, H15Q/S67L/I86T, I13V/H15Q/S67L/I86T, I13V/H15Q/E44D/V89D, I13V/S39I/E44D/Q82R/V89D, I13V/E44D/Q82R/V89D, I13V/Q72H/R76G/I86T, I13V/H15Q/R76G/I85F, H15Q/S39I/R76G/V89D, H15Q/S67L/R76G/I85F, H15Q/T47A/Q72H/R76G/I86T, H15Q/T47A/Q72H/R76G, I13V/H15Q/T47A/Q72H/R76G, H15Q/E44D/R76G/I85F, H15Q/S39I/S67L/V89D, H15Q/N32D/S67L/V89D, N32D/S67L/V89D, H15Q/S67L/Q72H/R76G/V89D, H15Q/Q72H/Q74R/R76G/I86T, G28V/Q72H/R76G/I86T, I13V/H15Q/S39I/E44D/S67L, E44D/S67L/Q72H/Q82R/V89D, H15Q/V89D, H15Q/T47A, I13V/H15Q/Q82R, I13V/H15Q/V89D, I13V/S67L/Q82R/V89D, I13V/H15Q/Q82R/V89D, H15Q/V31M/S67L/Q82R/V89D, I13V/H15Q/T47A/Q82R, I13V/H15Q/V31A/N45S/Q82R/V89D, H15Q/T47A/H69L/Q82R/V89D, I13V/H15Q/T47A/H69L/R76G/V89D, I12V/I13V/H15Q/T47A/Q82R/V89D, I13V/H15Q/R76G/D77N/Q82R/V89D, I13V/H15Q/T47A/R76G/V89D, I13V/H15Q/T47A/Q82R/V89D, I13V/H15Q/N24D/Q82R/V89D, I13V/H15Q/136V/T47A/S67L/V89D, H15Q/T47A/K65R/S67L/Q82R/V89D, H15Q/L33P/T47A/S67L/P71S/V89D, I13V/H15Q/Q72H/R76G/I86T, H15Q/T47A/S67L/Q82R/V89D, F2L/H15Q/D46E/T47A/Q72H/R76G/Q82R/V89D, I13V/H15Q/L33F/T47A/Q82R/V89D, I13V/H15Q/T47A/E58G/S67L/Q82R/V89D, H15Q/N24S/T47A/Q72H/R76G/V89D, I13V/H15Q/E44V/T47A/Q82R/V89D, H15Q/N18D/T47A/Q72H/V73A/R76G/I86T/V89D, I13V/H15Q/T37A/E44D/S48C/S67L/Q82R/V89D, H15Q/L33H/S67L/R76G/Q82R/V89D, I13V/H15Q/T47A/Q72H/R76G/I86T, H15Q/S39I/E44D/Q72H/V75G/R76G/Q82R/V89D, H15Q/T47A/S67L/R76G/Q82R/V89D, or I13V/H15Q/T47A/S67L/Q72H/R76G/Q82R/V89D.

In some of any such embodiments, the one or more amino acid substitutions correspond to position(s) selected from 13, 15, 39, 44, 47 67, 72, 76, 82, 85, 86, or 89. In some of any such embodiments, the one or more amino acid substitutions are selected from I13V, H15Q, E44D, T47A, S67L, Q72H, R76G, Q82R, I85F, I86T, V89D, or a conservative amino acid substitution thereof. In some of any such embodiments, the one or more amino acid substitutions correspond to position(s) selected from 13, 15, 44, 47, 67, 72, 76, 82, 86, or 89. In some of any such embodiments, the one or more amino acid substitutions are selected from I13V, H15Q, E44D, T47A, S67L, Q72H, R76G, Q82R, I86T, V89D, or a conservative amino acid substitution thereof.

In some embodiments, the variant PD-L2 polypeptide contains amino acid modifications I13V/H15Q, I13V/T47A, I13V/S67L, I13V/Q72H, I13V/Q72H, I13V/R76G, I13V/Q82R, I13V/I86T, I13V/V89D, H15Q/T47A, H15Q/S67L, H15Q/Q72H, H15Q/Q72H, H15Q/R76G, H15Q/Q82R, H15Q/I86T, H15Q/V89D, T47A/S67L, T47A/Q72H, T47A/Q72H, T47A/R76G, T47A/Q82R, T47A/I86T, T47A/V89D, S67L/Q72H, S67L/Q72H, S67L/R76G, S67L/Q82R, S67L/I86T, S67L/V89D, Q72H/R76G, Q72H/Q82R, Q72H/I86T, Q72H/V89D, R76G/Q82R, R76G/I86T, R76G/V89D, Q82R/I86T, Q82R/V89D or I86T/V89D. In some examples, the variant PD-L2 polypeptide contains amino acid modifications H15Q/S62L/Q82R, H15Q/S62L/V89D, H15Q/Q82R/V89D, or S62L/Q82R/V89D. In some instances, the variant PD-L2 polypeptide contains amino acid modifications H15Q/T47A/K65R/S67L/Q82R/V89D.

In some of any such embodiments, the variant PD-L2 polypeptide contains the IgV domain or a specific fragment thereof and the IgC domain or a specific fragment thereof. In some of any such embodiments, the variant PD-L2 polypeptide includes the sequence of amino acids set forth in any of SEQ ID NOS: 56-106, 108-114, 116-132 or a specific binding fragment thereof, or a sequence of amino acids that exhibits at least 95% sequence identity to any of SEQ ID NOS: 56-106, 108-114, 116-132 or a specific binding fragment thereof and that contains the one or more of the amino acid substitutions.

In some of any such embodiments, the variant PD-L2 polypeptide contains the IgV domain or a specific binding fragment thereof. In some of any such embodiments, the IgV domain or specific binding fragment thereof is the only PD-L2 portion of the variant PD-L2 polypeptide. In some of any such embodiments, the IgC domain or specific binding fragment thereof is the only PD-L2 portion of the variant PD-L2 polypeptide.

In some of any such embodiments, the variant PD-L2 polypeptide contains the sequence of amino acids set forth in any of SEQ ID NOS: 133-183, 185-191, 193-209, 268-318, 320-343 or a specific binding fragment thereof, a sequence of amino acids that exhibits at least 95% sequence identity to any of SEQ ID NOS: 133-183, 185-191, 193-209, 268-318, 320-343 or a specific binding fragment thereof and that contains the one or more of the amino acid substitutions. In some embodiments, the IgC domain or specific binding fragment thereof is the only PD-L2 portion of the variant PD-L2 polypeptide.

In some of any such embodiments, the variant PD-L2 polypeptide specifically binds to the ectodomain of PD-1 or RGMb with increased affinity compared to the binding of the unmodified PD-L2 to the ectodomain of PD-1 or RGMb. In some of any such embodiments, the variant PD-L2 polypeptide specifically binds to the ectodomain of PD-1 with increased affinity compared to binding of the unmodified PD-L2 to the ectodomain of PD-1. In some of any such embodiments, the variant PD-L2 polypeptide specifically binds to the ectodomain of PD-1 and the ectodomain of RGMb each with increased affinity compared to the binding of the unmodified PD-L2 to the ectodomain of PD-1 and the ectodomain of RGMb. In some of any such embodiments, the variant PD-L2 polypeptide specifically binds to the ectodomain of PD-1 with increased affinity and specifically binds to the ectodomain of the other of RGMb with decreased affinity compared to the binding of the unmodified PD-L2 to the ectodomain of the PD-1 or RGMb.

In some of any such embodiments, the increased affinity to the ectodomain of PD-1 is increased more than 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold or 60-fold compared to the unmodified PD-L2. In some embodiments, the increased affinity to the ectodomain of RGMb is increased more than 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold or 60-fold compared to the unmodified PD-L2. In some aspects, the decreased affinity to the ectodomain of RGMb is decreased more than 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold or 60-fold compared to the unmodified PD-L2.

In some of any such embodiments, the variant polypeptide specifically binds to the ectodomain of PD-1 with increased selectivity compared to the unmodified PD-L2. In some instances, the increased selectivity includes a greater ratio of binding of the variant polypeptide for PD-1 versus RGMb compared to the ratio of binding of the unmodified PD-L2 polypeptide for PD-1 versus RGMb. In some instances, the ratio is greater by at least or at least about 1.5-fold, 2.0-fold, 3.0-fold, 4.0-fold, 5.0-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold or more.

In some of any such embodiments, the PD-1 is a human PD-1. In some of any such embodiments, the RGMb is a human RGMb.

In some of any such embodiments, the binding activity is altered (increased or decreased) more than 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold 40-fold or 50-fold compared to the unmodified PD-L2.

In some of any such embodiments, the variant PD-L2 polypeptide is a soluble protein. In some embodiments, the variant PD-L2 polypeptide lacks the PD-L2 transmembrane domain and intracellular signaling domain; and/or the variant PD-L2 polypeptide is not capable of being expressed on the surface of a cell.

In some of any such embodiments, the variant PD-L2 polypeptide is linked to a multimerization domain. In some of any such embodiments, the variant PD-L2 polypeptide is a multimeric polypeptide, optionally a dimeric polypeptide, containing a first variant PD-L2 polypeptide linked to a multimerization domain and a second variant PD-L2 polypeptide linked to a multimerization domain. In some embodiments, the first variant PD-L2 polypeptide and the second variant PD-L2 polypeptide are the same or different.

In some of any such embodiments, the multimerization domain is an Fc domain or a variant thereof with reduced effector function. In some of any such embodiments, the variant PD-L2 polypeptide is linked to a moiety that increases biological half-life of the polypeptide. In some of any such embodiments, the variant PD-L2 polypeptide is linked to an Fc domain or a variant thereof with reduced effector function.

In some embodiments, the Fc domain is mammalian, optionally human; or the variant Fc domain contains one or more amino acid modifications compared to an unmodified Fc domain that is mammalian, optionally human. In some embodiments, the Fc domain or variant thereof contains the sequence of amino acids set forth in SEQ ID NO:211 or SEQ ID NO:212 or a sequence of amino acids that exhibits at least 85% sequence identity to SEQ ID NO:211 or SEQ ID NO:212. In some of any such embodiments, the Fc domain contains one or more amino acid modifications selected from among E233P, L234A, L234V, L235A, L235E, G236del, G237A, S267K, N297G, V302C, and K447del each by EU numbering. In some embodiments, the Fc domain contains the amino acid modification C220S by EU numbering. In some embodiments, the Fc domain contains the sequence of amino acids set forth in any of SEQ ID NOS: 1189, 1205, 1206, 1207, 1739, 1738, 1739, 1740 or a sequence of amino acids that exhibits at least 85% sequence identity to any of SEQ ID NOS: 1189, 1205, 1206, 1207, 1739, 1738, 1739, 1740 and exhibits reduced effector function.

In some of any such embodiments, the variant PD-L2 polypeptide is linked indirectly via a linker, optionally a G45 linker. In some of any such embodiments, the variant PD-L2 polypeptide is a transmembrane immunomodulatory protein further containing a transmembrane domain linked to the extracellular domain (ECD) or specific binding fragment thereof of the variant PD-L2 polypeptide. In some instances, the transmembrane domain contains the sequence of amino acids set forth as residues 221-241 of SEQ ID NO:4 or a functional variant thereof that exhibits at least 85% sequence identity to residues 221-241 of SEQ ID NO:4. In some embodiments, the variant PD-L2 polypeptide further contains a cytoplasmic signaling domain linked to the transmembrane domain. In some aspects, the cytoplasmic signaling domain includes the sequence of amino acids set forth as residues 242-273 of SEQ ID NO:4 or a functional variant thereof that exhibits at least 85% sequence identity to residues 242-273 of SEQ ID NO:4.

In some of any of the provided embodiments, the variant PD-L2 polypeptide modulates a response of an immune cell, such as a T cell. In some embodiments, the response, e.g. T cell response, is increased or is decreased. In some of any such embodiments, the variant PD-L2 increases IFN-gamma (interferon-gamma) expression relative to the unmodified PD-L2 in an in vitro T-cell assay. In some of any such embodiments, the variant PD-L2 decreases IFN-gamma (interferon-gamma) expression relative to the unmodified PD-L2 in an in vitro T-cell assay.

In some embodiments of any one of the variant PD-L2 polypeptides described herein, the variant PD-L2 polypeptide increases T cell signaling relative to the unmodified PD-L2 such as determined using a reporter assay involving a T cell (e.g. Jurkat) engineered with a reporter (e.g. luciferase) operably connected to an IL-2 promoter. In some embodiments of any one of the variant PD-L2 polypeptides described herein, the variant PD-L2 polypeptide decreases T cell signaling relative to the unmodified PD-L2, such as determined using a reporter assay involving a T cell (e.g. Jurkat) engineered with a reporter (e.g. luciferase) operably connected to an IL-2 promoter. In some of any such embodiments, the variant PD-L2 polypeptide is provided in any of a variety of formats, such as soluble or immobilized (e.g. plate-bound).

In some of any such embodiments, the variant PD-L2 polypeptide is deglycosylated.

Also provided is an immunomodulatory polypeptide containing the variant PD-L2 according to any of the provided embodiments linked, directly of indirectly via a linker, to a second polypeptide containing an immunoglobulin superfamily (IgSF) domain. In some cases, the IgSF domain is affinity modified and exhibits altered binding to one or more of its cognate binding partner(s) compared to the unmodified or wild-type IgSF domain. In some instances, the IgSF domain exhibits increased binding to one or more of its cognate binding partner(s) compared to the unmodified or wild-type IgSF domain to the same one or more cognate binding partner(s).

In some of any such embodiments, the variant PD-L2 is a first PD-L2 variant and the IgSF domain of the second polypeptide is an IgSF domain from a second variant PD-L2 of any of the provided embodiments, wherein the first and second PD-L2 variant are the same or different. In some embodiments, the variant PD-L2 polypeptide is capable of specifically binding to PD-1 or RGMb and the IgSF domain of the second polypeptide is capable of binding to a cognate binding partner other than one specifically bound by the PD-L2 variant polypeptide.

In some of any such embodiments, the IgSF domain is from a member of the B7 family. In some embodiments, the IgSF domain is a tumor-localizing moiety that binds to a ligand expressed on a tumor. In some cases, the ligand is B7H6. In some aspects, the IgSF domain is from NKp30.

In some of any such embodiments, the IgSF domain or affinity-modified IgSF domain thereof, optionally of the second or third polypeptide, is or contains an IgV domain. In some embodiments, the variant PD-L2 polypeptide is or contains an IgV domain. In some of any such embodiments, the immunomodulatory protein contains a multimerization domain linked to one or both of the variant PD-L2 polypeptide of the IgSF domain.

In some embodiments, the multimerization domain is an Fc domain or a variant thereof with reduced effector function. In some embodiments, the immunomodulatory protein is dimeric. In some cases, the immunomodulatory protein is homodimeric. In some instances, the immunomodulatory protein is heterodimeric.

In some of any such embodiments, the IgSF domain of the second polypeptide is an IgSF domain of a ligand that binds to an inhibitory receptor, or is an affinity-modified IgSF domain thereof. In some instances, the affinity-modified IgSF domain exhibits increased binding affinity and/or binding selectivity for the inhibitory receptor compared to binding of the unmodified IgSF domain to the same inhibitory receptor. In some embodiments, the inhibitory receptor is TIGIT or CTLA-4; or the ligand of the inhibitory receptor is CD155, CD112 or CD80.

In some of any such embodiments, the IgSF domain of the second polypeptide is an affinity-modified IgSF domain containing (i) a wildtype CD155 comprising an IgSF set forth in any of SEQ ID NOS: 47, 344, or 387, or a variant CD155 polypeptide comprising an IgSF domain of any of SEQ ID NOS set forth in Table 5, optionally any of the SEQ ID NOs: 345-386, 388-699, 1527-1736; (ii) a wildtype CD112 comprising an IgSF domain set forth in any of SEQ ID NOS: 48, 700, or 795, or a variant CD112 polypeptide comprising an IgSF domain of any of SEQ ID NOS set forth in Table 4, optionally any of the SEQ ID NOs: 701-794, 796-965, 1455-1526; (iii) a wildtype CD80 comprising an IgSF set forth in any of SEQ ID NOS:28, 1039, or 2039, or a variant CD80 polypeptide comprising an IgSF of any of SEQ ID NOS set forth in Table 3, optionally any of the SEQ ID NOs: 28, 966-998, 1000-1072, 1074-1146, 1147-1186; (iv) a wildtype PD-L1 comprising an IgSF set forth in any of SEQ ID NOS: 30, 1812, 1258, or 1454, or a variant PD-L1 polypeptide comprising an IgSF of any of SEQ ID NOS set forth in Table 8, optionally any of SEQ ID NOS: 1259-1453, 1743-1811, 1813-2021; (v) a sequence of amino acids that exhibits at least 95% sequence identity to any of the SEQ ID NOs in (i)-(iv) and that contains the amino acid substitution; or (vi) a specific binding fragment of any of (i)-(v).

In some embodiments, the immunomodulatory protein further contains a third polypeptide containing a wild-type IgSF domain or a variant or affinity-modified IgSF domain thereof, said affinity-modified IgSF domain comprising one or more amino acid modifications compared to the unmodified or wild-type IgSF domain of the IgSF family member, wherein the third polypeptide is the same as the first and/or second polypeptide; or the third polypeptide is different from the first and/or second polypeptide.

In some examples, the IgSF domain of the third polypeptide is an affinity-modified IgSF domain containing: (i) a wildtype CD155 comprising an IgSF set forth in any of SEQ ID NOS: 47, 344, or 387, or a variant CD155 polypeptide comprising an IgSF domain set forth in any of SEQ ID NOS: 345-386, 388-699, 1527-1736; (ii) a wildtype CD112 comprising an IgSF domain set forth in any of SEQ ID NOS: 48, 700, or 795, or a variant CD112 polypeptide comprising an IgSF domain set forth in any of SEQ ID NOS: 701-794, 796-965, 1455-1526; (iii) a wildtype CD80 comprising an IgSF set forth in any of SEQ ID NOS:28, 1039, or 2039, or a variant CD80 polypeptide comprising an IgSF domain set forth in any of SEQ ID NOS: 966-998, 1000-1038, 1040-1072, 1074-1112, 1114-1146, 1147-1186; (iv) a wildtype PD-L1 comprising an IgSF set forth in any of SEQ ID NOS: 30, 1812, 1258, or 1454, or a variant PD-L1 polypeptide comprising an IgSF domain set forth in any of SEQ ID NOS:1259-1453, 1743-1811, 1813-2021; (v) a sequence of amino acids that exhibits at least 95% sequence identity to any of the SEQ ID NOS in (i)-(iv) and that comprises the amino acid modifications, optionally amino acid substitutions, insertions and/or deletions thereof; or (vi) a specific binding fragment of any of (i)-(v).

In some of any such embodiments, the immunomodulatory protein further contains at least one additional polypeptide containing an IgSF domain of an IgSF family member or an affinity-modified IgSF domain thereof, said affinity-modified IgSF domain comprising one or more amino acid modifications compared to the binding of the unmodified or wild-type IgSF domain of the IgSF family member.

In some embodiments, the immunomodulatory protein further contains a multimerization domain linked to at least one of the variant PD-L2 polypeptide, the second polypeptide and/or the third polypeptide, optionally wherein the multimerization domain is an Fc domain or a variant thereof with reduced effector function.

In some embodiments, the multimerization domain promotes heterodimer formation.

Provided is an immunomodulatory protein containing a first variant PD-L2 polypeptide in which the multimerization domain is a first multimerization domain and a second variant PD-L2 polypeptide in which the multimerization domain is a second multimerization domain, wherein the first and second multimerization domains interact to form a multimer containing the first and second variant PD-L2 polypeptide, optionally wherein the first and second variant PD-L2 polypeptides are the same. Also provided is an immunomodulatory protein containing any of the provided immunomodulatory proteins, wherein the multimerization domain is a first multimerization domain and interacts with a second multimerization domain to form a multimer containing the immunomodulatory protein.

In some embodiments, the immunomodulatory protein is a first immunomodulatory protein and a second immunomodulatory protein is linked directly or indirectly via a linker to the second multimerization domain, wherein the multimer contains the first and second immunomodulatory protein. In some cases, the second immunomodulatory protein is an immunomodulatory protein as described herein and the multimerization domain is the second multimerization domain. In some embodiments, the multimer is a dimer. In some aspects, the immunomodulatory protein is a homodimer, optionally wherein the first and second multimerization domain is the same.

In some embodiments, the second polypeptide is a variant CD155 polypeptide and the first and/or second immunomodulatory protein includes the sequence set forth in any of SEQ ID NOS: 1191-1196, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to any of SEQ ID NOS: 1191-1196. In some cases, the immunomodulatory protein is a heterodimer, optionally wherein the first and second multimerization domain are different and/or are capable of interacting to mediate heterodimer formation. In some examples, the second polypeptide is a variant CD155 polypeptide and: the first or second immunomodulatory protein contains the sequence set forth in any of SEQ ID NOS: 1197, 1198, 1199, 1200, 1201, 1203 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to any of SEQ ID NOS: 1197, 1198, 1199, 1200, 1201 or 1203; and the other of the first or second immunomodulatory protein includes the sequence set forth in any of SEQ ID NOS: 1188, 1190, 1202 or 1204, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to any of SEQ ID NOS: 1188, 1190, 1202 or 1204.

In some embodiments, the first and/or second multimerization domain is an Fc domain or a variant thereof with reduced effector function, optionally wherein the Fc domain is of an immunoglobulin protein that is human and/or the Fc region is human, optionally wherein the Fc region is of an immunoglobulin G1 (IgG1) or an immunoglobulin G2 (IgG2), optionally set forth in SEQ ID NO:211 or SEQ ID NO:212; or the variant Fc domain contains one or more amino acid substitutions in a wildtype Fc region, optionally wherein the reduced effector function is reduced compared to a wildtype Fc region, optionally wherein the wildtype human Fc is of human IgG1.

In some of any such embodiments, the first and second multimerization domain is the same or different. In some examples, the variant Fc region contains the amino acid substitutions E233P, L234A, L234V, L235A, L235E, G236del, G237A, S267K, or N297G, with residue numbering according to the EU index of Kabat; or the amino acid substitutions R292C/N297G/V302C or L234A/L235E/G237A, with residue numbering according to the EU index of Kabat. In some cases, the Fc region or variant Fc region contains the amino acid substitution C220S, with residue numbering according to the EU index of Kabat. In some examples, the Fc region or variant Fc region contains K447del, with residue numbering according to the EU index of Kabat.

In some of any such embodiments, the sequence of amino acids set forth in any of SEQ ID NOS: 1191-1204 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to any of SEQ ID NOS: 1191-1204.

Also provided is a conjugate containing a variant PD-L2 according to any of the provided embodiments or an immunomodulatory polypeptide according to any of the provided embodiments linked to a moiety. In some cases, the moiety is a targeting moiety that specifically binds to a molecule on the surface of a cell. In some instances, the targeting moiety specifically binds to a molecule on the surface of an immune cell. In some embodiments, the immune cell is an antigen presenting cell or a lymphocyte.

In some cases, the targeting moiety is a tumor-localizing moiety that binds to a molecule on the surface of a tumor. In some of any such embodiments, the moiety is a protein, a peptide, nucleic acid, small molecule or nanoparticle. In some of any such embodiments, the moiety is an antibody or antigen-binding fragment. In some of any such embodiments, the conjugate is divalent, tetravalent, hexavalent or octavalent. In some cases, the conjugate is a fusion protein.

Also provided is a nucleic acid molecule(s) encoding a variant PD-L2 polypeptide according to any of the provided embodiments, a conjugate that is a fusion protein according to any of the provided embodiments, or an immunomodulatory polypeptide according to any of the provided embodiments. In some embodiments, the nucleic acid molecule is synthetic nucleic acid. In some instances, the nucleic acid molecule is cDNA.

Also provided is a vector containing the nucleic acid molecule according to any of the provided embodiments. In some cases, the vector is an expression vector. In some embodiments, the vector is a mammalian expression vector or a viral vector.

Also provided is a cell containing the vector of any of the provided embodiments. In some cases, the cell is a mammalian cell. In some aspect, the cell is a human cell.

Also provided is a method of producing a variant PD-L2 polypeptide or an immunomodulatory protein, including introducing the nucleic acid molecule according to any of the provided embodiments or vector according to any of the provided embodiments into a host cell under conditions to express the protein in the cell. In some cases, the method further includes isolating or purifying the variant PD-L2 polypeptide or immunomodulatory protein from the cell. Also provided is a method of engineering a cell expressing a variant PD-L2 variant polypeptide, including introducing a nucleic acid molecule encoding the variant PD-L2 polypeptide according to any of the provided embodiments into a host cell under conditions in which the polypeptide is expressed in the cell.

Also provided is an engineered cell, expressing the variant PD-L2 polypeptide according to any of the provided embodiments, a conjugate that is a fusion protein according to any of the provided embodiments, the immunomodulatory protein according to any of the provided embodiments, the nucleic acid molecule according to any of the provided embodiments, or the vector according to any of the embodiments.

In some cases the variant PD-L2 polypeptide or immunomodulatory protein contains a signal peptide. In some embodiments, the variant PD-L2 polypeptide or immunomodulatory protein does not contain a transmembrane domain and/or is not expressed on the surface of the cell. In some embodiments, the variant PD-L2 polypeptide or immunomodulatory protein is secreted or is capable of being secreted from the engineered cell. In some embodiments, the engineered cell contains a variant PD-L2 polypeptide that contains a transmembrane domain and/or is the transmembrane immunomodulatory protein according to any of the provided embodiments. In some embodiments, the variant PD-L2 polypeptide is expressed on the surface of the cell.

In some of any such embodiments, the cell is an immune cell. In some cases, the immune cell is an antigen presenting cell (APC) or a lymphocyte. In some embodiments, the engineered cell is a primary cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a lymphocyte and the lymphocyte is a T cell. In some cases, the cell is an APC and the APC is an artificial APC. In some examples, the engineered cell is a primary cell. In some cases, the cell is a mammalian cell. In some instances, the cell is a human cell. In some embodiments, the engineered cell further contains a chimeric antigen receptor (CAR) or an engineered T-cell receptor.

Also provided is an infectious agent, containing a nucleic acid molecule encoding a variant PD-L2 polypeptide according to any of the provided embodiments or an immunomodulatory polypeptide according to any of the provided embodiments. In some cases, the encoded variant PD-L2 polypeptide or immunomodulatory polypeptide does not contain a transmembrane domain and/or is not expressed on the surface of a cell in which it is expressed. In some embodiments, the encoded variant PD-L2 polypeptide or immunomodulatory polypeptide is secreted from a cell in which it is expressed. In some cases, the encoded variant PD-L2 polypeptide contains a transmembrane domain. In some embodiments, the encoded variant PD-L2 polypeptide is expressed on the surface of a cell in which it is expressed.

In some of any such embodiments, the infectious agent is a bacterium or a virus. In some embodiments, the virus is a lentiviral or retroviral construct or a hybrid thereof. In some instances, the virus is an oncolytic virus. In some examples, the oncolytic virus is an adenovirus, adeno-associated virus, herpes virus, Herpes Simplex Virus, Vesticular Stomatic virus, Reovirus, Newcastle Disease virus, parvovirus, measles virus, vesticular stomatitis virus (VSV), Coxsackie virus or a Vaccinia virus. In some aspects, the virus specifically targets dendritic cells (DCs) and/or is dendritic cell-tropic. In some embodiments, the virus is a lentiviral vector that is pseudotyped with a modified Sindbis virus envelope product.

In some embodiments, the infectious agent further contains a nucleic acid molecule encoding a further gene product that results in death of a target cell or that can augment or boost an immune response. In some cases, the further gene product is selected from an anticancer agent, anti-metastatic agent, an antiangiogenic agent, an immunomodulatory molecule, an immune checkpoint inhibitor, an antibody, a cytokine, a growth factor, an antigen, a cytotoxic gene product, a pro-apoptotic gene product, an anti-apoptotic gene product, a cell matrix degradative gene, genes for tissue regeneration or a reprogramming human somatic cells to pluripotency.

Also provided is a pharmaceutical composition containing the variant PD-L2 polypeptide according to any of the provided embodiments, an immunomodulatory protein according to any of the provided embodiments, a conjugate according to any of the provided embodiments, or an engineered cell according to any of the provided embodiments. In some instances, the pharmaceutical composition contains a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is sterile.

Also provided is an article of manufacture containing the pharmaceutical composition of according to any of the provided embodiments in a vial. In some cases, the vial is sealed.

Also provided is a kit containing the pharmaceutical composition according to any of the provided embodiments and instructions for use. Also provided is a kit containing the article of manufacture according to any of the provided embodiments and instructions for use.

Also provided is a method of modulating an immune response, such as increasing or decreasing an immune response in a subject including administering the pharmaceutical composition according to any of the provided embodiments to the subject. In some cases the method includes administering the engineered cells according to any of the provided embodiments. In some examples, the engineered cells are autologous to the subject. In some cases, the engineered cells are allogenic to the subject. In some embodiments, the method comprises administering to the subject a soluble variant PD-L2 polypeptide according to any one of the embodiments described herein, an immunomodulatory protein according to any one of the embodiments described herein or a conjugate according to any one of the embodiments described herein. In some embodiments, the method comprises administering to the subject an infectious agent encoding a variant PD-L2 polypeptide according to any one of the embodiments described herein.

In some embodiments, the method modulating the immune response treats a disease or condition in the subject.

In some of any such embodiments, the immune response is increased. Various formats of a variant PD-L2 polypeptide are contemplated for administration to a subject to increase an immune response, such as antagonist formats of a variant PD-L2. In some cases, such methods are carried out under conditions in which signaling by the inhibitory receptor PD-1 is blocked or attenuated by the administration. In some of any such embodiments, the method includes a variant PD-L2 polypeptide or immunomodulatory protein that is soluble is administered to the subject. In some instances, the soluble immunomodulatory protein is an immunomodulatory Fc fusion protein. In some embodiments of the method, a variant PD-L2 polypeptide according to any of the provided embodiments, or the immunomodulatory protein according to any of the provided embodiments is administered to the subject.

In some embodiments, an engineered cell containing a secretable variant PD-L2 polypeptide is administered to the subject. In some embodiments, an engineered cell according to any of the provided embodiments is administered to the subject. In some embodiments, an infectious agent encoding a variant PD-L2 polypeptide that is a secretable immunomodulatory protein is administered to the subject, optionally under conditions in which the infectious agent infects a tumor cell or immune cell and the secretable immunomodulatory protein is secreted from the infected cell.

In some of any such embodiments, the disease or condition is a tumor or cancer. In some embodiments, the disease or condition is selected from melanoma, lung cancer, bladder cancer, a hematological malignancy, liver cancer, brain cancer, renal cancer, breast cancer, pancreatic cancer, colorectal cancer, spleen cancer, prostate cancer, testicular cancer, ovarian cancer, uterine cancer, gastric carcinoma, a musculoskeletal cancer, a head and neck cancer, a gastrointestinal cancer, a germ cell cancer, or an endocrine and neuroendocrine cancer. In some of any such embodiments, the variant PD-L2 is administered in a format that increases an immune response in the subject.

In some of any such embodiments, the immune response is decreased. Various formats of a variant PD-L2 polypeptide are contemplated for administration to a subject to decrease an immune response, such as agonist formats of a variant PD-L2. In some cases, such methods are carried out under conditions in which signaling by the inhibitory receptor PD-1 is activated or stimulated or induced by the administration. In some embodiments, an immunomodulatory protein or conjugate comprising a variant PD-L2 polypeptide linked to an IgSF domain or a moiety that localizes to a cell or tissue of an inflammatory environment is administered to the subject. In some instances, the binding molecule contains an antibody or an antigen-binding fragment thereof or contains a wild-type IgSF domain or variant thereof.

In some embodiments, the immunomodulatory protein according to any of the provided embodiments or the conjugate according to any of the provided embodiments is administered to the subject. In some embodiments, a variant PD-L2 polypeptide that is a transmembrane immunomodulatory protein is administered to the subject. In some of any such embodiments, the engineered cell containing a variant PD-L2 polypeptide that is a transmembrane immunomodulatory protein according to any of the provided embodiments is administered to the subject. In some embodiments, an infectious agent encoding a variant PD-L2 polypeptide that is a transmembrane immunomodulatory protein is administered to the subject, optionally under conditions in which the infectious agent infects a tumor cell or immune cell and the transmembrane immunomodulatory protein is expressed on the surface of the infected cell.

In some of any such embodiments, the disease or condition is an inflammatory or autoimmune disease or condition. In some embodiments, the disease or condition is an antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis, a vasculitis, an autoimmune skin disease, transplantation, a Rheumatic disease, an inflammatory gastrointestinal disease, an inflammatory eye disease, an inflammatory neurological disease, an inflammatory pulmonary disease, an inflammatory endocrine disease, or an autoimmune hematological disease. In some embodiments, the disease or condition is selected from inflammatory bowel disease, transplant, Crohn's disease, ulcerative colitis, multiple sclerosis, asthma, rheumatoid arthritis, or psoriasis. In some of any such embodiments, the variant PD-L2 is administered in a format that decreases an immune response in the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1C depicts various formats of the provided variant IgSF domain molecules. FIG. 1A depicts soluble molecules, including: (1) a variant IgSF domain (vIgD) fused to an Fc chain; (2) a stack molecule containing a first variant IgSF domain (first vIgD) and a second IgSF domain, such as a second variant IgSF domain (second vIgD); (3) a tumor targeting IgSF molecule containing a first variant IgSF domain (vIgD) and an IgSF domain that targets to a tumor antigen, such as an NKp30 IgSF domain; and (4) a variant IgSF domain (vIgD) linked to an antibody (V-mAb). FIG. 1B depicts a transmembrane immunomodulatory protein (TIP) containing a variant IgSF domain (vIgD) expressed on the surface of a cell. In an exemplary embodiment, the cognate binding partner of the transmembrane bound vIgD is an inhibitory receptor (e.g. PD-1), and the TIP containing the vIgD (e.g. PD-L2 vIgD) antagonizes or blocks the negative signaling of the inhibitory receptor, thereby resulting in an activated T cell or effector T cell. In some cases, if clustering of the inhibitory receptor (PD-1) is proximal to an activating receptor (e.g. CD28) then agonizing activity by the TIP may be realized. FIG. 1C depicts a secreted immunomodulatory protein (SIP) in which a variant IgSF domain (vIgD) is secreted from a cell, such as a first T cell (e.g. CAR T cell). In an exemplary embodiment, the cognate binding partner of the secreted vIgD is an inhibitory receptor (e.g. PD-1), which can be expressed by the first cell (e.g. T cell, such as a CAR T cell) and/or on a second cell (e.g. T cell; either endogenous or engineered, such as a CAR T cell). Upon binding of the SIP with its cognate binding partner, the SIP antagonizes or blocks the negative signaling via the inhibitory receptor, thereby resulting in an activated T cell or effector T cell. In all cases, the vIgD can be a V-domain (IgV) only, the combination of the V-domain (IgV) and C-domain (IgC), including the entire extracellular domain (ECD), or any combination of Ig domains of the IgSF superfamily member.

FIG. 2 depicts an exemplary schematic of the activity of a variant IgSF domain (vIgD) fused to an Fc (vIgD-Fc) in which the vIgD is a variant of an IgSF domain of PD-L2. As shown, a soluble vIgD of PD-L2 interacts with its cognate binding partners to block interactions of PD-L1 or PD-L2 with PD-1, thereby blocking the PD-1 inhibitory receptor, and, in some cases, allowing the T cell to differentiate into an effector phenotype.

FIG. 3 depicts an exemplary schematic of a stack molecule that is a multi-target checkpoint antagonist containing a first variant IgSF domain (first vIgD) that is a PD-L1 or PD-L2 vIgD and a second IgSF domain (e.g. a second vIgD) that binds to a second inhibitory receptor. In the exemplary schematic, the second IgSF domain (e.g. second vIgD) is a CD112 or CD155 vIgD. As shown, the first vIgD and second vIgD interact with their cognate binding partners to block interactions of PD-L1 or PD-L2 with PD-1 and block interactions of CD155 or CD112 with TIGIT and/or CD112R, respectively, thereby blocking multiple inhibitory receptors.

FIG. 4 depicts an exemplary schematic of a stack molecule for localizing the variant IgSF (vIgD) to a tumor cell. In this format, the stack molecule contains a first variant IgSF domain (first vIgD) and a second IgSF domain (e.g. a second vIgD) in which the second IgSF domain (e.g a second vIgD) is a tumor-targeted IgSF domain that binds to a tumor antigen. An exemplary tumor-targeted IgSF domain is an IgSF domain of NKp30, which binds to the tumor antigen B7-H6. In this depiction, the first variant IgSF domain (vIgD) is a variant of an IgSF domain of PD-L2. As shown, binding of tumor-targeted IgSF domain to the surface of the tumor cell localizes the first variant IgSF domain on the tumor cell surface where it can interact with one or more of its cognate binding partners expressed on the surface of an adjacent immune cell (e.g. T cell) to antagonize inhibitory receptor signaling.

FIG. 5A depicts various exemplary configurations of a stack molecule containing a first variant IgSF domain (first vIgD) and a second IgSF domain, such as a second variant IgSF domain (second vIgD). As shown, the first vIgD and second IgSF domain are independently linked, directly or indirectly, to the N- or C-terminus of an Fc region. For generating a homodimeric Fc molecule, the Fc region is one that is capable of forming a homodimer with a matched Fc region by co-expression of the individual Fc regions in a cell. For generating a heterodimeric Fc molecule, the individual Fc regions contain mutations (e.g. “knob-into-hole” mutations in the CH3 domain), such that formation of the heterodimer is favored compared to homodimers when the individual Fc regions are co-expressed in a cell.

FIG. 5B depicts various exemplary configurations of a stack molecule containing a first variant IgSF domain (first vIgD), a second IgSF domain, such as a second variant IgSF domain (second vIgD), and a third IgSF domain, such as a third variant IgSF domain (third vIgD). As shown, the first vIgD, second IgSF, and third IgSF domains are independently linked, directly or indirectly, to the N- or C-terminus of an Fc region. For generating a homodimeric Fc molecule, the Fc region is one that is capable of forming a homodimer with a matched Fc region by co-expression of the individual Fc regions in a cell.

FIG. 6 depicts an exemplary schematic of the activity of a variant IgSF domain (vIgD)—conjugated to an antibody (V-Mab) in which the antibody (e.g. anti-HER2 antibody) binds to an antigen on the surface of the tumor cell to localize the vIgD to the cell. As shown, binding of the antibody to the surface of the tumor cell localizes the vIgD on the tumor cell surface where it can interact with one or more of its cognate binding partners expressed on the surface of an adjacent immune cell (e.g. T cell) to agonize or antagonize receptor signaling. In an exemplary embodiment as shown, the variant IgSF domain (vIgD) is a variant of an IgSF domain of PD-L2 that binds, such as has increased affinity for, the inhibitory receptor PD-1. Binding of the PD-L2 vIgD to the PD-1 inhibitory receptor antagonizes or blocks the negative signaling of the inhibitory receptor, thereby resulting in an activated T cell or effector T cell. In some cases, if clustering of the inhibitory receptor (PD-1) is proximal to an activating receptor (e.g. CD28) then agonizing of the inhibitory receptor activity by the TIP may be realized.

FIG. 7A-7C depicts various exemplary configurations of a variant IgSF-antibody conjugate (V-Mab). FIG. 7A shows various configurations in which a variant IgSF domain is linked, directly or indirectly, to the N- and/or C-terminus of the light chain of an antibody. FIG. 7B shows various configurations in which a variant IgSF domain is linked, directly or indirectly, to the N- and/or C-terminus of the heavy chain of an antibody. FIG. 7C depicts the results V-Mab configurations when a light chain of FIG. 7A and a heavy chain of FIG. 7B are co-expressed in a cell.

FIG. 8 depicts the MFI for binding to Jurakt/PD-1 cells at various concentrations of PD-L2 variant Fc fusion protein. Binding studies were carried out using Jurkat/IL-2 reporter cells that were then transduced to stably express PD-1 (Jurkat/PD-1). Cells were incubated with indicated concentrations of each candidate PD-L2 variant Fc fusion protein. As controls, a full extracellular domain of wild-type PD-L1 (“PDL1-FL”) and an IgV domain of wild-type PD-L2 (“wild type PD-L2 IgV”) or, anti-PD-1 monoclonal antibody (nivolumab), were tested. Unbound antibody was removed, bound antibody detected with fluorescently conjugated anti-human IgG, and the cells were analyzed by flow cytometry for MFI.

FIG. 9 and FIG. 10 depict the results for soluble variant PD-L2 IgV-Fc bioactivity tested in a human Mixed Lymphocyte Reaction (MLR). Approximately, 10,000 matured DC and 100,000 purified allogeneic CD4+ T cells were co-cultured with various increasing concentrations of variant PD-L2 IgV-Fc fusion proteins in 96 well round-bottom plates. Irrelevant human IgG or media only (designated “No Add”) were used as negative controls. As controls, either wildtype PDL2-Fc (full PD-L2 extracellular domain), wildtype PD-L2 IgV-Fc and or positive control anti-PD-1 monoclonal antibody (nivolumab) was assessed. IFN-gamma secretion in culture supernatants was analyzed and shown in FIG. 9 and FIG. 10.

FIG. 11A and FIG. 11B depicts the detection of PD-L2 SIP in supernatant of transduced CD19 CAR T cells and in HEK-293 cells, respectively.

FIG. 12A depicts the proliferation studies for T cells transduced with exemplary tested variant PD-L2 SIP. FIG. 12B depicts levels of IFN-gamma in the supernatant released by T cells transduced with exemplary tested variant PD-L2 SIP as measured by ELISA on day 5 after re-stimulation.

DETAILED DESCRIPTION

Provided herein are immunomodulatory proteins that are or comprise variants or mutants of Programmed cell death 1 ligand 2 (also known as PD-L2, PDCD1L2, PDCD1LG2, cluster of differentiation 273, CD273, or B7-DC) or specific binding fragments thereof that exhibit activity to bind to at least one target ligand cognate binding partner (also called counter-structure protein). In some embodiments, the variant PD-L2 polypeptides contain one or more amino acid modifications (e.g. amino acid substitutions, deletions or additions) compared to an unmodified or wild-type PD-L2 polypeptide. In some embodiments, the one or more amino acid modifications (e.g. substitutions) are in an IgSF domain (e.g. IgV) of an unmodified or wild-type PD-L2 polypeptide. In some embodiments, the variant PD-L2 polypeptide and immunomodulatory proteins exhibits altered, such as increased or decreased, binding activity or affinity for at least one cognate binding partner, such as at least one of PD-1 or RGMb. In some embodiments, the immunomodulatory proteins are soluble. In some embodiments, the immunomodulatory proteins are transmembrane immunomodulatory proteins capable of being expressed on the surface of cells. In some embodiments, also provided herein are one or more other immunomodulatory proteins that are conjugates or fusions containing a variant PD-L2 polypeptide provided herein and one or more other moiety or polypeptide.

In some embodiments, the variant PD-L2 polypeptides and immunomodulatory proteins modulate an immunological immune response, such as increase or decrease an immune response. In some embodiments, the variant PD-L2 polypeptides and immunomodulatory proteins provided herein can be used for the treatment of diseases or conditions that are associated with a dysregulated immune response.

In some embodiments, the provided variant PD-L2 polypeptides modulate T cell activation via interactions with costimulatory and/or coinhibitory signaling molecules. In general, antigen specific T-cell activation generally requires two distinct signals. The first signal is provided by the interaction of the T-cell receptor (TCR) with major histocompatibility complex (MHC) associated antigens present on antigen presenting cells (APCs). The second signal is costimulatory to TCR engagement and is necessary for T cell proliferation, differentiation and/or survival, including, in some cases, to avoid T-cell apoptosis or anergy.

In some embodiments, under normal physiological conditions, the T cell-mediated immune response is initiated by antigen recognition by the T cell receptor (TCR) and is regulated by a balance of co-stimulatory and inhibitory signals (e.g., immune checkpoint proteins). The immune system relies on immune checkpoints to prevent autoimmunity (i.e., self-tolerance) and to protect tissues from excessive damage during an immune response, for example during an attack against a pathogenic infection. In some cases, however, these immunomodulatory proteins can be dysregulated in diseases and conditions, including tumors, as a mechanism for evading the immune system.

In some embodiments, among known T-cell costimulatory receptors is Programmed cell death protein 1 or PD-1, which is the T-cell costimulatory receptor for the ligands PD-L1 (also known as cluster of differentiation 274, CD274. B7 homolog 1 or B7-H1) and PD-L2 (also known as PDCD1L2, PDCD1LG2, cluster of differentiation 273, CD273. or B7-DC). PD-L1 and PD-L2 are normally expressed on the surface of T cells, B cells, and myeloid cells. PD-L1 and PD-L2 are negative regulators of immune activation and are capable of down-modulating the immune response via interactions with programmed death 1 (PD-1) receptor. In some aspects, PD-1 is expressed on NK cells and T cells, including CD4+ and CD8+ T cells, whereby engagement of PD-1 can inhibit activation cell activation, proliferation, and/or expansion. However, PD-L2 ligands can also bind to Repulsive guidance molecule B or RGMb (also known as DRAGON or DRG11-responsive axonal guidance and outgrowth of neurite). The binding of PD-L2 to RGMb can block the interaction between PD-L2 and PD-1, and thereby potentiate or enhance the immune response. Thus, in some cases, interaction of PD-L2 with RGMb and PD-L2 with PD-1 yields opposing effects in modulating immune responses. Thus, PD-1 and RGMb may play opposing roles in immune responses to modulate pro-inflammatory or anti-inflammatory response, which, in some cases, are associated with a number of diseases and conditions.

In some embodiments, PD-1 and RGBb may play complementary roles in modeling an immune response. In some embodiments, enhancement or suppression of the activity of PD-1 receptor has clinical significance for treatment of inflammatory and autoimmune disorders, cancer, and viral infections. In some cases, however, therapies to intervene and alter the immunomodulatory effects of such receptors are constrained by the spatial orientation requirements as well as size limitations imposed by the confines of the immunological synapse. In some aspects, existing therapeutic drugs, including antibody drugs, may not be able to interact simultaneously with the multiple target proteins involved in modulating these interactions. In addition, in some cases, existing therapeutic drugs may only have the ability to antagonize but not agonize an immune response. Additionally, pharmacokinetic differences between drugs that independently target one of these receptors can create difficulties in properly maintaining a desired blood concentration of such drug combinations throughout the course of treatment.

In some embodiments, the provided variant PD-L2 polypeptides or immunomodulatory proteins modulate (e.g. increase or decrease) immunological activity associated PD-1. Thus, in some embodiments, the provided polypeptides overcome these constraints by providing variant PD-L2 with altered (e.g. increased or decreased) binding affinities to PD-1, thereby agonizing or antagonizing the effects of the receptor. In some embodiments, the provided polypeptides overcome these constraints by providing variant PD-L2 with altered (e.g. increased or decreased) binding affinities to RGMb, thereby modulating the effects of the interaction between PD-1 and PD-L2. Methods of making and using these variant PD-L2 are also provided.

All publications, including patents, patent applications scientific articles and databases, mentioned in this specification are herein incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, including patent, patent application, scientific article or database, were specifically and individually indicated to be incorporated by reference. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth herein prevails over the definition that is incorporated herein by reference.

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

I. DEFINITIONS

Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

The terms used throughout this specification are defined as follows unless otherwise limited in specific instances. As used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms, acronyms, and abbreviations used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Unless indicated otherwise, abbreviations and symbols for chemical and biochemical names is per IUPAC-IUB nomenclature. Unless indicated otherwise, all numerical ranges are inclusive of the values defining the range as well as all integer values in-between.

The term “affinity modified” as used in the context of an immunoglobulin superfamily domain, means a mammalian immunoglobulin superfamily (IgSF) domain having an altered amino acid sequence (relative to the corresponding wild-type parental or unmodified IgSF domain) such that it has an increased or decreased binding affinity or avidity to at least one of its cognate binding partners (alternatively “counter-structures”) compared to the parental wild-type or unmodified (i.e., non-affinity modified) IgSF control domain. Included in this context is an affinity modified PD-L2 IgSF domain. In some embodiments, the affinity-modified IgSF domain can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acid differences, such as amino acid substitutions, in a wildtype or unmodified IgSF domain. An increase or decrease in binding affinity or avidity can be determined using well known binding assays such as flow cytometry. Larsen et al., American Journal of Transplantation, Vol 5: 443-453 (2005). See also, Linsley et al., Immunity, Vol 1(9): 793-801 (1994). An increase in a protein's binding affinity or avidity to its cognate binding partner(s) is to a value at least 10% greater than that of the wild-type IgSF domain control and in some embodiments, at least 20%, 30%, 40%, 50%, 100%, 200%, 300%, 500%, 1000%, 5000%, or 10000% greater than that of the wild-type IgSF domain control value. A decrease in a protein's binding affinity or avidity to at least one of its cognate binding partner is to a value no greater than 90% of the control but no less than 10% of the wild-type IgSF domain control value, and in some embodiments no greater than 80%, 70% 60%, 50%, 40%, 30%, or 20% but no less than 10% of the wild-type IgSF domain control value. An affinity-modified protein is altered in primary amino acid sequence by substitution, addition, or deletion of amino acid residues. The term “affinity modified IgSF domain” is not to be construed as imposing any condition for any particular starting composition or method by which the affinity-modified IgSF domain was created. Thus, the affinity modified IgSF domains of the present invention are not limited to wild type IgSF domains that are then transformed to an affinity modified IgSF domain by any particular process of affinity modification. An affinity modified IgSF domain polypeptide can, for example, be generated starting from wild type mammalian IgSF domain sequence information, then modeled in silico for binding to its cognate binding partner, and finally recombinantly or chemically synthesized to yield the affinity modified IgSF domain composition of matter. In but one alternative example, an affinity modified IgSF domain can be created by site-directed mutagenesis of a wild-type IgSF domain. Thus, affinity modified IgSF domain denotes a product and not necessarily a product produced by any given process. A variety of techniques including recombinant methods, chemical synthesis, or combinations thereof, may be employed.

The term “allogeneic” as used herein means a cell or tissue that is removed from one organism and then infused or adoptively transferred into a genetically dissimilar organism of the same species. In some embodiments of the invention, the species is murine or human.

The term “autologous” as used herein means a cell or tissue that is removed from the same organism to which it is later infused or adoptively transferred. An autologous cell or tissue can be altered by, for example, recombinant DNA methodologies, such that it is no longer genetically identical to the native cell or native tissue which is removed from the organism. For example, a native autologous T-cell can be genetically engineered by recombinant DNA techniques to become an autologous engineered cell expressing a transmembrane immunomodulatory protein and/or chimeric antigen receptor (CAR), which in some cases involves engineering a T-cell or TIL (tumor infiltrating lymphocyte). The engineered cells are then infused into a patient from which the native T-cell was isolated. In some embodiments, the organism is human or murine.

The terms “binding affinity,” and “binding avidity” as used herein means the specific binding affinity and specific binding avidity, respectively, of a protein for its counter-structure under specific binding conditions. In biochemical kinetics avidity refers to the accumulated strength of multiple affinities of individual non-covalent binding interactions, such as between PD-L2 and its counter-structures PD-1 and/or RGMb. As such, avidity is distinct from affinity, which describes the strength of a single interaction. An increase or attenuation in binding affinity of a variant PD-L2 containing an affinity modified PD-L2 IgSF domain to its counter-structure is determined relative to the binding affinity of the unmodified PD-L2, such as an unmodified PD-L2 containing the native or wild-type IgSF domain, such as IgV domain. Methods for determining binding affinity or avidity are known in art. See, for example, Larsen et al., American Journal of Transplantation, Vol 5: 443-453 (2005). In some embodiments, a variant PD-L2 of the invention (i.e. a PD-L2 protein containing an affinity modified IgSF domain) specifically binds to PD-1 and/or RGMb measured by flow cytometry with a binding affinity that yields a Mean Fluorescence Intensity (MFI) value at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% greater than a wild-type PD-L2 control in a binding assay.

The term “biological half-life” refers to the amount of time it takes for a substance, such as an immunomodulatory polypeptide comprising a variant PD-L2 of the present invention, to lose half of its pharmacologic or physiologic activity or concentration. Biological half-life can be affected by elimination, excretion, degradation (e.g., enzymatic) of the substance, or absorption and concentration in certain organs or tissues of the body. In some embodiments, biological half-life can be assessed by determining the time it takes for the blood plasma concentration of the substance to reach half its steady state level (“plasma half-life”). Conjugates that can be used to derivatize and increase the biological half-life of polypeptides of the invention are known in the art and include, but are not limited to, polyethylene glycol (PEG), hydroxyethyl starch (HES), XTEN (extended recombinant peptides; see, WO2013130683), human serum albumin (HSA), bovine serum albumin (BSA), lipids (acylation), and poly-Pro-Ala-Ser (PAS), polyglutamic acid (glutamylation).

The term “chimeric antigen receptor” or “CAR” as used herein refers to an artificial (i.e., man-made) transmembrane protein expressed on a mammalian cell comprising at least an ectodomain, a transmembrane, and an endodomain. Optionally, the CAR protein includes a “spacer” which covalently links the ectodomain to the transmembrane domain. A spacer is often a polypeptide linking the ectodomain to the transmembrane domain via peptide bonds. The CAR is typically expressed on a mammalian lymphocyte. In some embodiments, the CAR is expressed on a mammalian cell such as a T-cell or a tumor infiltrating lymphocyte (TIL). A CAR expressed on a T-cell is referred to herein as a “CAR T-cell” or “CAR-T.” In some embodiments the CAR-T is a T helper cell, a cytotoxic T-cell, a natural killer T-cell, a memory T-cell, a regulatory T-cell, or a gamma delta T-cell. When used clinically in, e.g. adoptive cell transfer, a CAR-T with antigen binding specificity to the patient's tumor is typically engineered to express on a T-cell obtained from the patient. The engineered T-cell expressing the CAR is then infused back into the patient. The CAR-T is thus often an autologous CAR-T although allogeneic CAR-T are included within the scope of the invention. The ectodomain of a CAR comprises an antigen binding region, such as an antibody or antigen binding fragment thereof (e.g. scFv), that specifically binds under physiological conditions with a target antigen, such as a tumor specific antigen. Upon specific binding a biochemical chain of events (i.e., signal transduction) results in modulation of the immunological activity of the CAR-T. Thus, for example, upon specific binding by the antigen binding region of the CAR-T to its target antigen can lead to changes in the immunological activity of the T-cell activity as reflected by changes in cytotoxicity, proliferation or cytokine production. Signal transduction upon CAR-T activation is achieved in some embodiments by the CD3-zeta chain (“CD3-z”) which is involved in signal transduction in native mammalian T-cells. CAR-Ts can further comprise multiple signaling domains such as CD28, 41BB or OX40, to further modulate immunomodulatory response of the T-cell. CD3-z comprises a conserved motif known as an immunoreceptor tyrosine-based activation motif (ITAM) which is involved in T-cell receptor signal transduction.

The term “collectively” or “collective” when used in reference to cytokine production induced by the presence of two or more variant PD-L2 of the invention in an in vitro assay, means the overall cytokine expression level irrespective of the cytokine production induced by individual variant PD-L2. In some embodiments, the cytokine being assayed is IFN-gamma, such as in an in vitro primary T-cell assay.

The term “cognate binding partner” (used interchangeably with “counter-structure”) in reference to a polypeptide, such as in reference to an IgSF domain of a variant PD-L2, refers to at least one molecule (typically a native mammalian protein) to which the referenced polypeptide specifically binds under specific binding conditions. In some aspects, a variant PD-L2 containing an affinity modified IgSF domain specifically binds to the counter-structure of the corresponding native or wildtype PD-L2 but with increased or attenuated affinity. A species of ligand recognized and specifically binding to its cognate receptor under specific binding conditions is an example of a counter-structure or cognate binding partner of that receptor. A “cognate cell surface binding partner” is a cognate binding partner expressed on a mammalian cell surface. A “cell surface molecular species” is a cognate binding partner of ligands of the immunological synapse (IS), expressed on and by cells, such as mammalian cells, forming the immunological synapse.

As used herein, “conjugate,” “conjugation” or grammatical variations thereof refers the joining or linking together of two or more compounds resulting in the formation of another compound, by any joining or linking methods known in the art. It can also refer to a compound which is generated by the joining or linking together two or more compounds. For example, a variant PD-L2 polypeptide linked directly or indirectly to one or more chemical moieties or polypeptide is an exemplary conjugate. Such conjugates include fusion proteins, those produced by chemical conjugates and those produced by any other methods.

The term “competitive binding” as used herein means that a protein is capable of specifically binding to at least two cognate binding partners but that specific binding of one cognate binding partner inhibits, such as prevents or precludes, simultaneous binding of the second cognate binding partner. Thus, in some cases, it is not possible for a protein to bind the two cognate binding partners at the same time. Generally, competitive binders contain the same or overlapping binding site for specific binding but this is not a requirement. In some embodiments, competitive binding causes a measurable inhibition (partial or complete) of specific binding of a protein to one of its cognate binding partner due to specific binding of a second cognate binding partner. A variety of methods are known to quantify competitive binding such as ELISA (enzyme linked immunosorbent assay) assays.

The term “conservative amino acid substitution” as used herein means an amino acid substitution in which an amino acid residue is substituted by another amino acid residue having a side chain R group with similar chemical properties (e.g., charge or hydrophobicity). Examples of groups of amino acids that have side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid; and 7) sulfur-containing side chains: cysteine and methionine. Conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine.

The term, “corresponding to” with reference to positions of a protein, such as recitation that nucleotides or amino acid positions “correspond to” nucleotides or amino acid positions in a disclosed sequence, such as set forth in the Sequence listing, refers to nucleotides or amino acid positions identified upon alignment with the disclosed sequence based on structural sequence alignment or using a standard alignment algorithm, such as the GAP algorithm. For example, corresponding residues can be determined by alignment of a reference sequence with the sequence of wild-type PD-L2 set forth in SEQ ID NO:31 (ECD domain) or set forth in SEQ ID NO: 55 (IgV domain) by structural alignment methods as described herein. By aligning the sequences, one skilled in the art can identify corresponding residues, for example, using conserved and identical amino acid residues as guides.

The terms “decrease” or “attenuate” “or suppress” as used herein means to decrease by a statistically significant amount. A decrease can be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

The terms “derivatives” or “derivatized” refer to modification of a protein by covalently linking it, directly or indirectly, to a composition so as to alter such characteristics as biological half-life, bioavailability, immunogenicity, solubility, toxicity, potency, or efficacy while retaining or enhancing its therapeutic benefit. Derivatives of immunomodulatory polypeptides of the invention are within the scope of the invention and can be made by, for example, glycosylation, pegylation, lipidation, or Fc-fusion.

As used herein, “domain” (typically a sequence of three or more, generally 5 or 7 or more amino acids, such as 10 to 200 amino acid residues) refers to a portion of a molecule, such as a protein or encoding nucleic acid, that is structurally and/or functionally distinct from other portions of the molecule and is identifiable. For example, domains include those portions of a polypeptide chain that can form an independently folded structure within a protein made up of one or more structural motifs and/or that is recognized by virtue of a functional activity, such as binding activity. A protein can have one, or more than one, distinct domains. For example, a domain can be identified, defined or distinguished by homology of the primary sequence or structure to related family members, such as homology to motifs. In another example, a domain can be distinguished by its function, such as an ability to interact with a biomolecule, such as a cognate binding partner. A domain independently can exhibit a biological function or activity such that the domain independently or fused to another molecule can perform an activity, such as, for example binding. A domain can be a linear sequence of amino acids or a non-linear sequence of amino acids. Many polypeptides contain a plurality of domains. Such domains are known, and can be identified by those of skill in the art. For exemplification herein, definitions are provided, but it is understood that it is well within the skill in the art to recognize particular domains by name. If needed appropriate software can be employed to identify domains.

The term “ectodomain” as used herein refers to the region of a membrane protein, such as a transmembrane protein, that lies outside the vesicular membrane. Ectodomains often comprise binding domains that specifically bind to ligands or cell surface receptors, such as via a binding domain that specifically binds to the ligand or cell surface receptor. The ectodomain of a cellular transmembrane protein is alternately referred to as an extracellular domain.

The terms “effective amount” or “therapeutically effective amount” refer to a quantity and/or concentration of a therapeutic composition of the invention, including a protein composition or cell composition, that when administered ex vivo (by contact with a cell from a patient) or in vivo (by administration into a patient) either alone (i.e., as a monotherapy) or in combination with additional therapeutic agents, yields a statistically significant decrease in disease progression as, for example, by ameliorating or eliminating symptoms and/or the cause of the disease. An effective amount may be an amount that relieves, lessens, or alleviates at least one symptom or biological response or effect associated with a disease or disorder, prevents progression of the disease or disorder, or improves physical functioning of the patient. In the case of cell therapy, the effective amount is an effective dose or number of cells administered to a patient by adoptive cell therapy. In some embodiments the patient is a mammal such as a non-human primate or human patient.

The term “endodomain” as used herein refers to the region found in some membrane proteins, such as transmembrane proteins, that extends into the interior space defined by the cell surface membrane. In mammalian cells, the endodomain is the cytoplasmic region of the membrane protein. In cells, the endodomain interacts with intracellular constituents and can be play a role in signal transduction and thus, in some cases, can be an intracellular signaling domain. The endodomain of a cellular transmembrane protein is alternately referred to as a cytoplasmic domain, which, in some cases, can be a cytoplasmic signaling domain.

The terms “enhanced” or “increased” as used herein in the context of increasing immunological activity of a mammalian lymphocyte means to increase one or more activities the lymphocyte. An increased activity can be one or more of increase cell survival, cell proliferation, cytokine production, or T-cell cytotoxicity, such as by a statistically significant amount. In some embodiments, reference to increased immunological activity means to increase interferon gamma (IFN-gamma) production, such as by a statistically significant amount. In some embodiments, the immunological activity can be assessed in a mixed lymphocyte reaction (MLR) assay. Methods of conducting MLR assays are known in the art. Wang et al., Cancer Immunol Res. 2014 September: 2(9):846-56. Other methods of assessing activities of lymphocytes are known in the art, including any assay as described herein. In some embodiments an enhancement can be an increase of at least 10%, 20%, 30%, 40%, 50%, 75%, 100%, 200%, 300%, 400%, or 500% greater than a non-zero control value.

The term “engineered cell” as used herein refers to a mammalian cell that has been genetically modified by human intervention such as by recombinant DNA methods or viral transduction. In some embodiments, the cell is an immune cell, such as a lymphocyte (e.g. T cell, B cell, NK cell) or an antigen presenting cell (e.g. dendritic cell). The cell can be a primary cell from a patient or can be a cell line. In some embodiments, an engineered cell of the invention comprises a variant PD-L2 of the invention engineered to modulate immunological activity of a T-cell expressing PD-1 and/or RGMb to which the variant PD-L2 specifically binds. In some embodiments, the variant PD-L2 is a transmembrane immunomodulatory protein (hereinafter referred to as “TIP”) containing the extracellular domain or a portion thereof containing the IgV domain linked to a transmembrane domain (e.g. a PD-L2 transmembrane domain) and, optionally, an intracellular signaling domain. In some cases, the TIP is formatted as a chimeric receptor containing a heterologous cytoplasmic signaling domain or endodomain. In some embodiments, an engineered cell is capable of expressing and secreting an immunomodulatory protein as described herein. Among provided engineered cells also are cells further containing an engineered T-cell receptor (TCR) or chimeric antigen receptor (CAR).

The term “engineered T-cell” as used herein refers to a T-cell such as a T helper cell, cytotoxic T-cell (alternatively, cytotoxic T lymphocyte or CTL), natural killer T-cell, regulatory T-cell, memory T-cell, or gamma delta T-cell, that has been genetically modified by human intervention such as by recombinant DNA methods or viral transduction methods. An engineered T-cell comprises a variant PD-L2 transmembrane immunomodulatory protein (TIP) or secreted immunodulatory protein (SIP) of the present invention that is expressed on the T-cell and is engineered to modulate immunological activity of the engineered T-cell itself, or a mammalian cell to which the variant PD-L2 expressed on the T-cell specifically binds.

The term “engineered T-cell receptor” or “engineered TCR” refers to a T-cell receptor (TCR) engineered to specifically bind with a desired affinity to a major histocompatibility complex (MHC)/peptide target antigen that is selected, cloned, and/or subsequently introduced into a population of T-cells, often used for adoptive immunotherapy. In contrast to engineered TCRs, CARs are engineered to bind target antigens in a MHC independent manner.

The term “expressed on” as used herein is used in reference to a protein expressed on the surface of a cell, such as a mammalian cell. Thus, the protein is expressed as a membrane protein. In some embodiments, the expressed protein is a transmembrane protein. In some embodiments, the protein is conjugated to a small molecule moiety such as a drug or detectable label. Proteins expressed on the surface of a cell can include cell-surface proteins such as cell surface receptors that are expressed on mammalian cells.

The term “half-life extending moiety” refers to a moiety of a polypeptide fusion or chemical conjugate that extends the half-life of a protein circulating in mammalian blood serum compared to the half-life of the protein that is not so conjugated to the moiety. In some embodiments, half-life is extended by greater than or greater than about 1.2-fold, 1.5-fold, 2.0-fold, 3.0-fold, 4.0-fold, 5.0-fold, or 6.0-fold. In some embodiments, half-life is extended by more than 6 hours, more than 12 hours, more than 24 hours, more than 48 hours, more than 72 hours, more than 96 hours or more than 1 week after in vivo administration compared to the protein without the half-life extending moiety. The half-life refers to the amount of time it takes for the protein to lose half of its concentration, amount, or activity. Half-life can be determined for example, by using an ELISA assay or an activity assay. Exemplary half-life extending moieties include an Fc domain, a multimerization domain, polyethylene glycol (PEG), hydroxyethyl starch (HES), XTEN (extended recombinant peptides; see, WO2013130683), human serum albumin (HSA), bovine serum albumin (BSA), lipids (acylation), and poly-Pro-Ala-Ser (PAS), and polyglutamic acid (glutamylation).

The term “immunological synapse” or “immune synapse” as used herein means the interface between a mammalian cell that expresses MHC I (major histocompatibility complex) or MHC II, such as an antigen-presenting cell or tumor cell, and a mammalian lymphocyte such as an effector T cell or Natural Killer (NK) cell.

An Fc (fragment crystallizable) region or domain of an immunoglobulin molecule (also termed an Fc polypeptide) corresponds largely to the constant region of the immunoglobulin heavy chain, and is responsible for various functions, including the antibody's effector function(s). The Fc domain contains part or all of a hinge domain of an immunoglobulin molecule plus a CH2 and a CH3 domain. The Fc domain can form a dimer of two polypeptide chains joined by one or more disulfide bonds. In some embodiments, the Fc is a variant Fc that exhibits reduced (e.g. reduced greater than 30%, 40%, 50%, 60%, 70%, 80%, 90% or more) activity to facilitate an effector function. In some embodiments, reference to amino acid substitutions in an Fc region is by EU numbering system unless described with reference to a specific SEQ ID NO. EU numbering is known and is according to the most recently updated IMGT Scientific Chart (IMGT®, the international ImMunoGeneTics information System®, http://www.imgt.org/IMGTScientificChart/Numbering/Hu_IGHGnber.html (created: 17 May 2001, last updated: 10 Jan. 2013) and the EU index as reported in Kabat, E. A. et al. Sequences of Proteins of Immunological interest. 5th ed. US Department of Health and Human Services, NIH publication No. 91-3242 (1991).

An immunoglobulin Fc fusion (“Fc-fusion”), such as an immunomodulatory Fc fusion protein, is a molecule comprising one or more polypeptides (or one or more small molecules) operably linked to an Fc region of an immunoglobulin. An Fc-fusion may comprise, for example, the Fc region of an antibody (which facilitates effector functions and pharmacokinetics) and a variant PD-L2. An immunoglobulin Fc region may be linked indirectly or directly to one or more variant PD-L2 or small molecules (fusion partners). Various linkers are known in the art and can optionally be used to link an Fc to a fusion partner to generate an Fc-fusion. Fc-fusions of identical species can be dimerized to form Fc-fusion homodimers, or using non-identical species to form Fc-fusion heterodimers. In some embodiments, the Fc is a mammalian Fc such as a murine or human Fc.

The term “host cell” refers to a cell that can be used to express a protein encoded by a recombinant expression vector. A host cell can be a prokaryote, for example, E. coli, or it can be a eukaryote, for example, a single-celled eukaryote (e.g., a yeast or other fungus), a plant cell (e.g., a tobacco or tomato plant cell), an animal cell (e.g., a human cell, a monkey cell, a hamster cell, a rat cell, a mouse cell, or an insect cell) or a hybridoma. Examples of host cells include Chinese hamster ovary (CHO) cells or their derivatives such as Veggie CHO and related cell lines which grow in serum-free media or CHO strain DX-B11, which is deficient in DHFR. Another example is Human Endothelial Kidney 293 cells or their derivatives. In some embodiments, a host cell is a mammalian cell (e.g., a human cell, a monkey cell, a hamster cell, a rat cell, a mouse cell, or an insect cell).

The term “immunoglobulin” (abbreviated “Ig”) as used herein refers to a mammalian immunoglobulin protein including any of the five human classes of antibody: IgA (which includes subclasses IgA1 and IgA2), IgD, IgE, IgG (which includes subclasses IgG1, IgG2, IgG3, and IgG4), and IgM. The term is also inclusive of immunoglobulins that are less than full-length, whether wholly or partially synthetic (e.g., recombinant or chemical synthesis) or naturally produced, such as antigen binding fragment (Fab), variable fragment (Fv) containing V_(H) and V_(L), the single chain variable fragment (scFv) containing V_(H) and V_(L) linked together in one chain, as well as other antibody V region fragments, such as Fab′, F(ab)₂, F(ab′)₂, dsFv diabody, Fc, and Fd polypeptide fragments. Bispecific antibodies, homobispecific and heterobispecific, are included within the meaning of the term.

The term “immunoglobulin superfamily” or “IgSF” as used herein means the group of cell surface and soluble proteins that are involved in the recognition, binding, or adhesion processes of cells. Molecules are categorized as members of this superfamily based on shared structural features with immunoglobulins (i.e., antibodies); they all possess a domain known as an immunoglobulin domain or fold. Members of the IgSF include cell surface antigen receptors, co-receptors and co-stimulatory molecules of the immune system, molecules involved in antigen presentation to lymphocytes, cell adhesion molecules, certain cytokine receptors and intracellular muscle proteins. They are commonly associated with roles in the immune system. Proteins in the immunological synapse are often members of the IgSF. IgSF can also be classified into “subfamilies” based on shared properties such as function. Such subfamilies typically consist of from 4 to 30 IgSF members.

The terms “IgSF domain” or “immunoglobulin domain” or “Ig domain” as used herein refers to a structural domain of IgSF proteins. Ig domains are named after the immunoglobulin molecules. They contain about 70-110 amino acids and are categorized according to their size and function. Ig-domains possess a characteristic Ig-fold, which has a sandwich-like structure formed by two sheets of antiparallel beta strands. Interactions between hydrophobic amino acids on the inner side of the sandwich and highly conserved disulfide bonds formed between cysteine residues in the B and F strands, stabilize the Ig-fold. One end of the Ig domain has a section called the complementarity determining region that is important for the specificity of antibodies for their ligands. The Ig like domains can be classified (into classes) as: IgV, IgC (which either can be an IgC1 or IgC2), or IgI. Most Ig domains are either variable (IgV) or constant (IgC). IgV domains with 9 beta strands are generally longer than IgC domains with 7 beta strands. Ig domains of some members of the IgSF resemble IgV domains in the amino acid sequence, yet are similar in size to IgC domains. These are called IgC2 domains, while standard IgC domains are called IgC1 domains. T-cell receptor (TCR) chains contain two Ig domains in the extracellular portion; one IgV domain at the N-terminus and one IgC1 domain adjacent to the cell membrane. PD-L2 contains two Ig domains: one IgV and one IgC domain.

The term “IgSF species” as used herein means an ensemble of IgSF member proteins with identical or substantially identical primary amino acid sequence. Each mammalian immunoglobulin superfamily (IgSF) member defines a unique identity of all IgSF species that belong to that IgSF member. Thus, each IgSF family member is unique from other IgSF family members and, accordingly, each species of a particular IgSF family member is unique from the species of another IgSF family member. Nevertheless, variation between molecules that are of the same IgSF species may occur owing to differences in post-translational modification such as glycosylation, phosphorylation, ubiquitination, nitrosylation, methylation, acetylation, and lipidation. Additionally, minor sequence differences within a single IgSF species owing to gene polymorphisms constitute another form of variation within a single IgSF species as do wild type truncated forms of IgSF species owing to, for example, proteolytic cleavage. A “cell surface IgSF species” is an IgSF species expressed on the surface of a cell, generally a mammalian cell.

The term “immunological activity” as used herein in the context of mammalian lymphocytes such as T-cells refers to one or more cell survival, cell proliferation, cytokine production (e.g. interferon-gamma), or T-cell cytotoxicity activities. In some cases, an immunological activity can mean the cell expression of cytokines, such as chemokines or interleukins. Assays for determining enhancement or suppression of immunological activity include the MLR (mixed lymphocyte reaction) assays measuring interferon-gamma cytokine levels in culture supernatants (Wang et al., Cancer Immunol Res. 2014 September: 2(9):846-56), SEB (staphylococcal enterotoxin B) T cell stimulation assay (Wang et al., Cancer Immunol Res. 2014 September: 2(9):846-56), and anti-CD3 T cell stimulation assays (Li and Kurlander, J Transl Med. 2010: 8: 104). Since T cell activation is associated with secretion of IFN-gamma cytokine, detecting IFN-gamma levels in culture supernatants from these in vitro human T cell assays can be assayed using commercial ELISA kits (Wu et al, Immunol Lett 2008 Apr. 15; 117(1): 57-62). Induction of an immune response results in an increase in immunological activity relative to quiescent lymphocytes. An immunomodulatory protein, such as a variant PD-L2 polypeptide containing an affinity modified IgSF domain, as provided herein can in some embodiments increase or, in alternative embodiments, decrease IFN-gamma (interferon-gamma) expression in a primary T-cell assay relative to a wild-type IgSF member or IgSF domain control. Those of skill will recognize that the format of the primary T-cell assay used to determine an increase in IFN-gamma expression can differ from that employed to assay for a decrease in IFN-gamma expression. In assaying for the ability of an immunomodulatory protein or affinity modified IgSF domain of the invention to alter IFN-gamma expression in a primary T-cell assay, a Mixed Lymphocyte Reaction (MLR) assay can be used. Conveniently, in some cases, a soluble form of an affinity modified IgSF domain of the invention can be employed to determine its ability to increase or decrease the IFN-gamma expression in a MLR. Alternatively, a co-immobilization assay can be used. In a co-immobilization assay, a T-cell receptor signal, provided in some embodiments by anti-CD3 antibody, is used in conjunction with a co-immobilized affinity modified IgSF domain, such as variant PD-L2, to determine the ability to increase or decrease IFN-gamma expression relative to a wild-type IgSF domain control. Methods to assay the immunological activity of engineered cells, including to evaluate the activity of a variant PD-L2 transmembrane immunomodulatory protein, are known in the art and include, but are not limited to, the ability to expand T cells following antigen stimulation, sustain T cell expansion in the absence of re-stimulation, and anti-cancer activities in appropriate animal models. Assays also include assays to assess cytotoxicity, including a standard ⁵¹Cr-release assay (see e.g. Milone et al., (2009) Molecular Therapy 17: 1453-1464) or flow based cytotoxicity assays, or an impedance based cytotoxicity assay (Peper et al. (2014) Journal of Immunological Methods, 405:192-198).

An “immunomodulatory polypeptide” or “immunomodulatory protein” is a polypeptide or protein molecule that modulates immunological activity. By “modulation” or “modulating” an immune response is meant that immunological activity is either increased or decreased. An immunomodulatory protein can be a single polypeptide chain or a multimer (dimers or higher order multimers) of at least two polypeptide chains covalently bonded to each other by, for example, interchain disulfide bonds. Thus, monomeric, dimeric, and higher order multimeric polypeptides are within the scope of the defined term. Multimeric polypeptides can be homomultimeric (of identical polypeptide chains) or heteromultimeric (of non-identical polypeptide chains). An immunomodulatory protein of the invention comprises a variant PD-L2.

The term “increase” as used herein means to increase by a statistically significant amount. An increase can be at least 5%, 10%, 20%, 30%, 40%, 50%, 75%, 100%, or greater than a non-zero control value.

An “isoform” of PD-L2 is one of a plurality naturally occurring PD-L2 polypeptides that differ in amino acid sequence. Isoforms can be the product of splice variants of an RNA transcript expressed by a single gene, or the expression product of highly similar but different genes yielding a functionally similar protein such as may occur from gene duplication. As used herein, the term “isoform” of PD-L2 also refers to the product of different alleles of a PD-L2 gene.

The term “lymphocyte” as used herein means any of three subtypes of white blood cell in a mammalian immune system. They include natural killer cells (NK cells) (which function in cell-mediated, cytotoxic innate immunity), T cells (for cell-mediated, cytotoxic adaptive immunity), and B cells (for humoral, antibody-driven adaptive immunity). T cells include: T helper cells, cytotoxic T-cells, natural killer T-cells, memory T-cells, regulatory T-cells, or gamma delta T-cells. Innate lymphoid cells (ILC) are also included within the definition of lymphocyte.

The terms “mammal,” or “patient” specifically includes reference to at least one of a: human, chimpanzee, rhesus monkey, cynomolgus monkey, dog, cat, mouse, or rat.

The term “membrane protein” as used herein means a protein that, under physiological conditions, is attached directly or indirectly to a lipid bilayer. A lipid bilayer that forms a membrane can be a biological membrane such as a eukaryotic (e.g., mammalian) cell membrane or an artificial (i.e., man-made) membrane such as that found on a liposome. Attachment of a membrane protein to the lipid bilayer can be by way of covalent attachment, or by way of non-covalent interactions such as hydrophobic or electrostatic interactions. A membrane protein can be an integral membrane protein or a peripheral membrane protein. Membrane proteins that are peripheral membrane proteins are non-covalently attached to the lipid bilayer or non-covalently attached to an integral membrane protein. A peripheral membrane protein forms a temporary attachment to the lipid bilayer such that under the range of conditions that are physiological in a mammal, peripheral membrane protein can associate and/or disassociate from the lipid bilayer. In contrast to peripheral membrane proteins, integral membrane proteins form a substantially permanent attachment to the membrane's lipid bilayer such that under the range of conditions that are physiological in a mammal, integral membrane proteins do not disassociate from their attachment to the lipid bilayer. A membrane protein can form an attachment to the membrane by way of one layer of the lipid bilayer (monotopic), or attached by way of both layers of the membrane (polytopic). An integral membrane protein that interacts with only one lipid bilayer is an “integral monotopic protein”. An integral membrane protein that interacts with both lipid bilayers is an “integral polytopic protein” alternatively referred to herein as a “transmembrane protein”.

The terms “modulating” or “modulate” as used herein in the context of an immune response, such as a mammalian immune response, refer to any alteration, such as an increase or a decrease, of existing or potential immune responses that occurs as a result of administration of an immunomodulatory polypeptide comprising a variant PD-L2 of the present invention or as a result of administration of engineered cells expresses an immunomodulatory protein, such as a variant PD-L2 transmembrane immunomodulatory protein of the present invention. Thus, it refers to an alteration, such as an increase or decrease, of an immune response as compared to the immune response that occurs or is present in the absence of the administration of the immunomodulatory protein comprising the variant PD-L2 or cells expressing such an immunomodulatory polypeptide. Such modulation includes any induction, activation, suppression or alteration in degree or extent of immunological activity of an immune cell. Immune cells include B cells, T cells, NK (natural killer) cells, NK T cells, professional antigen-presenting cells (APCs), and non-professional antigen-presenting cells, and inflammatory cells (neutrophils, macrophages, monocytes, eosinophils, and basophils). Modulation includes any change imparted on an existing immune response, a developing immune response, a potential immune response, or the capacity to induce, regulate, influence, or respond to an immune response. Modulation includes any alteration in the expression and/or function of genes, proteins and/or other molecules in immune cells as part of an immune response. Modulation of an immune response or modulation of immunological activity includes, for example, the following: elimination, deletion, or sequestration of immune cells; induction or generation of immune cells that can modulate the functional capacity of other cells such as autoreactive lymphocytes, antigen presenting cells, or inflammatory cells; induction of an unresponsive state in immune cells (i.e., anergy); enhancing or suppressing the activity or function of immune cells, including but not limited to altering the pattern of proteins expressed by these cells. Examples include altered production and/or secretion of certain classes of molecules such as cytokines, chemokines, growth factors, transcription factors, kinases, costimulatory molecules, or other cell surface receptors or any combination of these modulatory events. Modulation can be assessed, for example, by an alteration in IFN-gamma (interferon gamma) expression relative to the wild-type PD-L2 control in a primary T cell assay (see, Zhao and Ji, Exp Cell Res. 2016 Jan. 1; 340(1) 132-138). Modulation can be assessed, for example, by an alteration of an immunological activity of engineered cells, such as an alteration in in cytotoxic activity of engineered cells or an alteration in cytokine secretion of engineered cells relative to cells engineered with a wild-type PD-L2 transmembrane protein

The term “molecular species” as used herein means an ensemble of proteins with identical or substantially identical primary amino acid sequence. Each mammalian immunoglobulin superfamily (IgSF) member defines a collection of identical or substantially identical molecular species. Thus, for example, human PD-L2 is an IgSF member and each human PD-L2 molecule is a molecular species of PD-L2. Variation between molecules that are of the same molecular species may occur owing to differences in post-translational modification such as glycosylation, phosphorylation, ubiquitination, nitrosylation, methylation, acetylation, and lipidation. Additionally, minor sequence differences within a single molecular species owing to gene polymorphisms constitute another form of variation within a single molecular species as do wild type truncated forms of a single molecular species owing to, for example, proteolytic cleavage. A “cell surface molecular species” is a molecular species expressed on the surface of a mammalian cell. Two or more different species of protein, each of which is present exclusively on one or exclusively the other (but not both) of the two mammalian cells forming the IS, are said to be in “cis” or “cis configuration” with each other. Two different species of protein, the first of which is exclusively present on one of the two mammalian cells forming the IS and the second of which is present exclusively on the second of the two mammalian cells forming the IS, are said to be in “trans” or “trans configuration.” Two different species of protein each of which is present on both of the two mammalian cells forming the IS are in both cis and trans configurations on these cells.

The term, a “multimerization domain” refers to a sequence of amino acids that promotes stable interaction of a polypeptide molecule with one or more additional polypeptide molecules, each containing a complementary multimerization domain (e.g. a first multimerization domain and a second multimerization domain), which can be the same or a different multimerization domain. The interactions between complementary multimerization domains, e.g. interaction between a first multimerization domain and a second multimerization domain, form a stable protein-protein interaction to produce a multimer of the polypeptide molecule with the additional polypeptide molecule. In some cases, the multimerization domain is the same and interacts with itself to form a stable protein-protein interaction between two polypeptide chains. Generally, a polypeptide is joined directly or indirectly to the multimerization domain. Exemplary multimerization domains include the immunoglobulin sequences or portions thereof, leucine zippers, hydrophobic regions, hydrophilic regions, and compatible protein-protein interaction domains. The multimerization domain, for example, can be an immunoglobulin constant region or domain, such as, for example, the Fc domain or portions thereof from IgG, including IgG1, IgG2, IgG3 or IgG4 subtypes, IgA, IgE, IgD and IgM and modified forms thereof.

The terms “nucleic acid” and “polynucleotide” are used interchangeably to refer to a polymer of nucleic acid residues (e.g., deoxyribonucleotides or ribonucleotides) in either single- or double-stranded form. Unless specifically limited, the terms encompass nucleic acids containing known analogues of natural nucleotides and that have similar binding properties to it and are metabolized in a manner similar to naturally-occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary nucleotide sequences as well as the sequence explicitly indicated (a “reference sequence”). Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues. The term nucleic acid or polynucleotide encompasses cDNA or mRNA encoded by a gene.

The term “non-competitive binding” as used herein means the ability of a protein to specifically bind simultaneously to at least two cognate binding partners. Thus, the protein is able to bind to at least two different cognate binding partners at the same time, although the binding interaction need not be for the same duration such that, in some cases, the protein is specifically bound to only one of the cognate binding partners. In some embodiments, the binding occurs under specific binding conditions. In some embodiments, the simultaneous binding is such that binding of one cognate binding partner does not substantially inhibit simultaneous binding to a second cognate binding partner. In some embodiments, non-competitive binding means that binding a second cognate binding partner to its binding site on the protein does not displace the binding of a first cognate binding partner to its binding site on the protein. Methods of assessing non-competitive binding are well known in the art such as the method described in Perez de La Lastra et al., Immunology, 1999 April: 96(4): 663-670. In some cases, in non-competitive interactions, the first cognate binding partner specifically binds at an interaction site that does not overlap with the interaction site of the second cognate binding partner such that binding of the second cognate binding partner does not directly interfere with the binding of the first cognate binding partner. Thus, any effect on binding of the cognate binding partner by the binding of the second cognate binding partner is through a mechanism other than direct interference with the binding of the first cognate binding partner. For example, in the context of enzyme-substrate interactions, a non-competitive inhibitor binds to a site other than the active site of the enzyme. Non-competitive binding encompasses uncompetitive binding interactions in which a second cognate binding partner specifically binds at an interaction site that does not overlap with the binding of the first cognate binding partner but binds to the second interaction site only when the first interaction site is occupied by the first cognate binding partner.

The term “pharmaceutical composition” refers to a composition suitable for pharmaceutical use in a mammalian subject, often a human. A pharmaceutical composition typically comprises an effective amount of an active agent (e.g., an immunomodulatory polypeptide comprising a variant PD-L2 or engineered cells expressing a variant PD-L2 transmembrane immunomodulatory protein) and a carrier, excipient, or diluent. The carrier, excipient, or diluent is typically a pharmaceutically acceptable carrier, excipient or diluent, respectively.

The terms “polypeptide” and “protein” are used interchangeably herein and refer to a molecular chain of two or more amino acids linked through peptide bonds. The terms do not refer to a specific length of the product. Thus, “peptides,” and “oligopeptides,” are included within the definition of polypeptide. The terms include post-translational modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like. The terms also include molecules in which one or more amino acid analogs or non-canonical or unnatural amino acids are included as can be synthesized, or expressed recombinantly using known protein engineering techniques. In addition, proteins can be derivatized.

The term “primary T-cell assay” as used herein refers to an in vitro assay to measure interferon-gamma (“IFN-gamma”) expression. A variety of such primary T-cell assays are known in the art. In a preferred embodiment, the assay used is an anti-CD3 coimmobilizaton assay. In this assay, primary T cells are stimulated by anti-CD3 immobilized with or without additional recombinant proteins. Culture supernatants are harvested at timepoints, usually 24-72 hours. In another embodiment, the assay used is the MLR. In this assay, primary T cells are stimulated with allogeneic APC. Culture supernatants are harvested at timepoints, usually 24-72 hours. Human IFN-gamma levels are measured in culture supernatants by standard ELISA techniques. Commercial kits are available from vendors and the assay is performed according to manufacturer's recommendation.

The term “purified” as applied to nucleic acids, such as encoding immunomodulatory proteins of the invention, generally denotes a nucleic acid or polypeptide that is substantially free from other components as determined by analytical techniques well known in the art (e.g., a purified polypeptide or polynucleotide forms a discrete band in an electrophoretic gel, chromatographic eluate, and/or a media subjected to density gradient centrifugation). For example, a nucleic acid or polypeptide that gives rise to essentially one band in an electrophoretic gel is “purified.” A purified nucleic acid or protein of the invention is at least about 50% pure, usually at least about 75%, 80%, 85%, 90%, 95%, 96%, 99% or more pure (e.g., percent by weight or on a molar basis).

The term “recombinant” indicates that the material (e.g., a nucleic acid or a polypeptide) has been artificially (i.e., non-naturally) altered by human intervention. The alteration can be performed on the material within, or removed from, its natural environment or state. For example, a “recombinant nucleic acid” is one that is made by recombining nucleic acids, e.g., during cloning, affinity modification, DNA shuffling or other well-known molecular biological procedures. A “recombinant DNA molecule,” is comprised of segments of DNA joined together by means of such molecular biological techniques. The term “recombinant protein” or “recombinant polypeptide” as used herein refers to a protein molecule which is expressed using a recombinant DNA molecule. A “recombinant host cell” is a cell that contains and/or expresses a recombinant nucleic acid or that is otherwise altered by genetic engineering, such as by introducing into the cell a nucleic acid molecule encoding a recombinant protein, such as a transmembrane immunomodulatory protein provided herein. Transcriptional control signals in eukaryotes comprise “promoter” and “enhancer” elements. Promoters and enhancers consist of short arrays of DNA sequences that interact specifically with cellular proteins involved in transcription. Promoter and enhancer elements have been isolated from a variety of eukaryotic sources including genes in yeast, insect and mammalian cells and viruses (analogous control elements, i.e., promoters, are also found in prokaryotes). The selection of a particular promoter and enhancer depends on what cell type is to be used to express the protein of interest. The terms “in operable combination,” “in operable order” and “operably linked” as used herein refer to the linkage of nucleic acid sequences in such a manner or orientation that a nucleic acid molecule capable of directing the transcription of a given gene and/or the synthesis of a desired protein molecule is produced.

The term “recombinant expression vector” as used herein refers to a DNA molecule containing a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host cell. Nucleic acid sequences necessary for expression in prokaryotes include a promoter, optionally an operator sequence, a ribosome binding site and possibly other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals. A secretory signal peptide sequence can also, optionally, be encoded by the recombinant expression vector, operably linked to the coding sequence for the recombinant protein, such as a recombinant fusion protein, so that the expressed fusion protein can be secreted by the recombinant host cell, for easier isolation of the fusion protein from the cell, if desired. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Among the vectors are viral vectors, such as lentiviral vectors.

The term “selectivity” refers to the preference of a subject protein, or polypeptide, for specific binding of one substrate, such as one cognate binding partner, compared to specific binding for another substrate, such as a different cognate binding partner of the subject protein. Selectivity can be reflected as a ratio of the binding activity (e.g. binding affinity) of a subject protein and a first substrate, such as a first cognate binding partner, (e.g., K_(d1)) and the binding activity (e.g. binding affinity) of the same subject protein with a second cognate binding partner (e.g., K_(d2)).

The term “sequence identity” as used herein refers to the sequence identity between genes or proteins at the nucleotide or amino acid level, respectively. “Sequence identity” is a measure of identity between proteins at the amino acid level and a measure of identity between nucleic acids at nucleotide level. The protein sequence identity may be determined by comparing the amino acid sequence in a given position in each sequence when the sequences are aligned. Similarly, the nucleic acid sequence identity may be determined by comparing the nucleotide sequence in a given position in each sequence when the sequences are aligned. Methods for the alignment of sequences for comparison are well known in the art, such methods include GAP, BESTFIT, BLAST, FASTA and TFASTA. The BLAST algorithm calculates percent sequence identity and performs a statistical analysis of the similarity between the two sequences. The software for performing BLAST analysis is publicly available through the National Center for Biotechnology information (NCBI) website.

The term “soluble” as used herein in reference to proteins, means that the protein is not a membrane protein. In general, a soluble protein contains only the extracellular domain of an IgSF family member receptor, or a portion thereof containing an IgSF domain or domains or specific-binding fragments thereof, but does not contain the transmembrane domain. In some cases, solubility of a protein can be improved by linkage or attachment, directly or indirectly via a linker, to an Fc domain, which, in some cases, also can improve the stability and/or half-life of the protein. In some aspects, a soluble protein is an Fc fusion protein.

The term “species” as used herein with respect to polypeptides or nucleic acids means an ensemble of molecules with identical or substantially identical sequences. Variation between polypeptides that are of the same species may occur owing to differences in post-translational modification such as glycosylation, phosphorylation, ubiquitination, nitrosylation, methylation, acetylation, and lipidation. Slightly truncated sequences of polypeptides that differ (or encode a difference) from the full length species at the amino-terminus or carboxy-terminus by no more than 1, 2, or 3 amino acid residues are considered to be of a single species. Such microheterogeneities are a common feature of manufactured proteins.

The term “specific binding fragment” as used herein in reference to a full-length wild-type mammalian PD-L2 polypeptide or an IgV or an IgC (e.g. IgC2) domain thereof, means a polypeptide having a subsequence of the full-length polypeptide or an IgV and/or IgC domain and that specifically binds in vitro and/or in vivo to a mammalian PD-1 and/or mammalian RGMb such as a human or murine PD-1 or RGMb. In some embodiments, the specific binding fragment comprises an PD-L2 IgV or a PD-L2 IgC2 subsequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% the sequence length of the full-length wild-type sequence or an IgV or a an IgC (e.g. IgC2) sequence thereof. The specific binding fragment can be altered in sequence to form a variant PD-L2 of the invention.

The term “specifically binds” as used herein means the ability of a protein, under specific binding conditions, to bind to a target protein such that its affinity or avidity is at least 5 times as great, but optionally at least 10, 20, 30, 40, 50, 100, 250 or 500 times as great, or even at least 1000 times as great as the average affinity or avidity of the same protein to a collection of random peptides or polypeptides of sufficient statistical size. A specifically binding protein need not bind exclusively to a single target molecule but may specifically bind to a non-target molecule due to similarity in structural conformation between the target and non-target (e.g., paralogs or orthologs). Those of skill will recognize that specific binding to a molecule having the same function in a different species of animal (i.e., ortholog) or to a non-target molecule having a substantially similar epitope as the target molecule (e.g., paralog) is possible and does not detract from the specificity of binding which is determined relative to a statistically valid collection of unique non-targets (e.g., random polypeptides). Thus, a polypeptide of the invention may specifically bind to more than one distinct species of target molecule due to cross-reactivity. Solid-phase ELISA immunoassays, ForteBio Octet, or Biacore measurements can be used to determine specific binding between two proteins. Generally, interactions between two binding proteins have dissociation constants (K_(d)) less than 1×10⁻⁵ M, and often as low as 1×10⁻¹² M. In certain embodiments of the present disclosure, interactions between two binding proteins have dissociation constants of less than or less than about 1×10⁻⁶ M, 1×10⁻⁷ M, 1×10⁻⁸ M, 1×10⁻⁹ M, 1×10⁻¹⁰ M or 1×10⁻¹¹M or less.

The terms “surface expresses” or “surface expression” in reference to a mammalian cell expressing a polypeptide means that the polypeptide is expressed as a membrane protein. In some embodiments, the membrane protein is a transmembrane protein.

As used herein, “synthetic,” with reference to, for example, a synthetic nucleic acid molecule or a synthetic gene or a synthetic peptide refers to a nucleic acid molecule or polypeptide molecule that is produced by recombinant methods and/or by chemical synthesis methods.

The term “targeting moiety” as used herein refers to a composition that is covalently or non-covalently attached to, or physically encapsulates, a polypeptide comprising a variant PD-L2 of the present invention. The targeting moiety has specific binding affinity for a desired counter-structure such as a cell surface receptor (e.g., PD-1), or a tumor antigen such as tumor specific antigen (TSA) or a tumor associated antigen (TAA) such as B7-H6. Typically, the desired counter-structure is localized on a specific tissue or cell-type. Targeting moieties include: antibodies, antigen binding fragment (Fab), variable fragment (Fv) containing V_(H) and V_(L), the single chain variable fragment (scFv) containing V_(H) and V_(L) linked together in one chain, as well as other antibody V region fragments, such as Fab′, F(ab)₂, F(ab′)₂, dsFv diabody, nanobodies, soluble receptors, receptor ligands, affinity matured receptors or ligands, as well as small molecule (<500 dalton) compositions (e.g., specific binding receptor compositions). Targeting moieties can also be attached covalently or non-covalently to the lipid membrane of liposomes that encapsulate a polypeptide of the present invention.

The term “transmembrane protein” as used herein means a membrane protein that substantially or completely spans a lipid bilayer such as those lipid bilayers found in a biological membrane such as a mammalian cell, or in an artificial construct such as a liposome. The transmembrane protein comprises a transmembrane domain (“transmembrane domain”) by which it is integrated into the lipid bilayer and by which the integration is thermodynamically stable under physiological conditions. Transmembrane domains are generally predictable from their amino acid sequence via any number of commercially available bioinformatics software applications on the basis of their elevated hydrophobicity relative to regions of the protein that interact with aqueous environments (e.g., cytosol, extracellular fluid). A transmembrane domain is often a hydrophobic alpha helix that spans the membrane. A transmembrane protein can pass through the both layers of the lipid bilayer once or multiple times. A transmembrane protein includes the provided transmembrane immunomodulatory proteins described herein. In addition to the transmembrane domain, a transmembrane immunomodulatory protein of the invention further comprises an ectodomain and, in some embodiments, an endodomain.

The terms “treating,” “treatment,” or “therapy” of a disease or disorder as used herein mean slowing, stopping or reversing the disease or disorders progression, as evidenced by decreasing, cessation or elimination of either clinical or diagnostic symptoms, by administration of a therapeutic composition (e.g. containing an immunomodulatory protein or engineered cells) of the invention either alone or in combination with another compound as described herein. “Treating,” “treatment,” or “therapy” also means a decrease in the severity of symptoms in an acute or chronic disease or disorder or a decrease in the relapse rate as for example in the case of a relapsing or remitting autoimmune disease course or a decrease in inflammation in the case of an inflammatory aspect of an autoimmune disease. As used herein in the context of cancer, the terms “treatment” or, “inhibit,” “inhibiting” or “inhibition” of cancer refers to at least one of: a statistically significant decrease in the rate of tumor growth, a cessation of tumor growth, or a reduction in the size, mass, metabolic activity, or volume of the tumor, as measured by standard criteria such as, but not limited to, the Response Evaluation Criteria for Solid Tumors (RECIST), or a statistically significant increase in progression free survival (PFS) or overall survival (OS). “Preventing,” “prophylaxis,” or “prevention” of a disease or disorder as used in the context of this invention refers to the administration of an immunomodulatory polypeptide or engineered cells of the invention, either alone or in combination with another compound, to prevent the occurrence or onset of a disease or disorder or some or all of the symptoms of a disease or disorder or to lessen the likelihood of the onset of a disease or disorder.

The term “tumor specific antigen” or “TSA” as used herein refers to a counter-structure that is present primarily on tumor cells of a mammalian subject but generally not found on normal cells of the mammalian subject. A tumor specific antigen need not be exclusive to tumor cells but the percentage of cells of a particular mammal that have the tumor specific antigen is sufficiently high or the levels of the tumor specific antigen on the surface of the tumor are sufficiently high such that it can be targeted by anti-tumor therapeutics, such as immunomodulatory polypeptides of the invention, and provide prevention or treatment of the mammal from the effects of the tumor. In some embodiments, in a random statistical sample of cells from a mammal with a tumor, at least 50% of the cells displaying a TSA are cancerous. In other embodiments, at least 60%, 70%, 80%, 85%, 90%, 95%, or 99% of the cells displaying a TSA are cancerous.

The term “variant” (also “modified” or mutant”) as used in reference to a variant PD-L2 means a PD-L2, such as a mammalian (e.g., human or murine) PD-L2 created by human intervention. The variant PD-L2 is a polypeptide having an altered amino acid sequence, relative to an unmodified or wild-type PD-L2. The variant PD-L2 is a polypeptide which differs from a wild-type PD-L2 isoform sequence by one or more amino acid substitutions, deletions, additions, or combinations thereof. For purposes herein, the variant PD-L2 contains at least one affinity modified domain, whereby one or more of the amino acid differences occurs in an IgSF domain (e.g. IgV domain). A variant PD-L2 can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acid differences, such as amino acid substitutions. A variant PD-L2 polypeptide generally exhibits at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a corresponding wild-type or unmodified PD-L2, such as to the sequence of SEQ ID NO:3, a mature sequence thereof (lacking the signal sequence) or a portion thereof containing the extracellular domain or an IgSF domain thereof. In some embodiments, a variant PD-L2 polypeptide exhibits at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a corresponding wild-type or unmodified PD-L2 comprising the sequence set forth in SEQ ID NO:31 or SEQ ID NO: 55. Non-naturally occurring amino acids as well as naturally occurring amino acids are included within the scope of permissible substitutions or additions. A variant PD-L2 is not limited to any particular method of making and includes, for example, de novo chemical synthesis, de novo recombinant DNA techniques, or combinations thereof. A variant PD-L2 of the invention specifically binds to at least one or more of PD-1 or RGMb of a mammalian species. In some embodiments, the altered amino acid sequence results in an altered (i.e., increased or decreased) binding affinity or avidity to PD-1 and/or RGMb compared to the wild-type or unmodified PD-L2 protein. An increase or decrease in binding affinity or avidity can be determined using well known binding assays such as flow cytometry. Larsen et al., American Journal of Transplantation, Vol 5: 443-453 (2005). See also, Linsley et al., Immunity, Vol 1(9): 793-801 (1994). An increase in variant PD-L2 binding affinity or avidity to PD-1 and/or RGMb is to a value at least 5% greater than that of the wild-type or unmodified PD-L2 and in some embodiments, at least 10%, 15%, 20%, 30%, 40%, 50%, 100% greater than that of the wild-type or unmodified PD-L2 control value. A decrease in PD-L2 binding affinity or avidity to PD-1 and/or RGMb is to a value no greater than 95% of the wild-type or unmodified control values, and in some embodiments no greater than 80%, 70% 60%, 50%, 40%, 30%, 20%, 10%, 5%, or no detectable binding affinity or avidity of the wild-type or unmodified control values. A variant PD-L2 is altered in primary amino acid sequence by substitution, addition, or deletion of amino acid residues. The term “variant” in the context of variant PD-L2 is not to be construed as imposing any condition for any particular starting composition or method by which the variant PD-L2 is created. A variant PD-L2 can, for example, be generated starting from wild type mammalian PD-L2 sequence information, then modeled in silico for binding to PD-1 and/or RGMb, and finally recombinantly or chemically synthesized to yield a variant PD-L2 of the present invention. In but one alternative example, a variant PD-L2 can be created by site-directed mutagenesis of a wild-type PD-L2. Thus, variant PD-L2 denotes a composition and not necessarily a product produced by any given process. A variety of techniques including recombinant methods, chemical synthesis, or combinations thereof, may be employed.

The term “wild-type” or “natural” or “native” as used herein is used in connection with biological materials such as nucleic acid molecules, proteins (e.g., PD-L2), IgSF members, host cells, and the like, refers to those which are found in nature and not modified by human intervention.

II. VARIANT PD-L2 POLYPEPTIDES

Provided herein are variant PD-L2 polypeptides that exhibit altered (increased or decreased) binding activity or affinity for one or more of a PD-L2 cognate binding partner. In some embodiments, the PD-L2 cognate binding partner is PD-1 or RGMb. In some embodiments, the PD-L2 cognate binding partner is PD-1. In some embodiments, the variant PD-L2 polypeptide contains one or more amino acids modifications, such as one or more substitutions (alternatively, “mutations” or “replacements”), deletions or addition, in an immunoglobulin superfamily (IgSF) domain (IgD) relative to a wild-type or unmodified PD-L2 polypeptide or a portion of a wild-type or unmodified PD-L2 containing an immunoglobulin superfamily (IgSF) domain or a specific binding fragment thereof. Thus, a provided variant PD-L1 polypeptide is or comprises a variant IgD (hereinafter called “vIgD”) in which the one or more amino acid modifications (e.g. substitutions) is in an IgD.

In some embodiments, the IgD comprises an IgV domain or an IgC (e.g. IgC2) domain or specific binding fragment of the IgV domain or the IgC (e.g. IgC2) domain, or combinations thereof. In some embodiments, the IgD can be an IgV only, the combination of the IgV and IgC, including the entire extracellular domain (ECD), or any combination of Ig domains of PD-L2. Table 2 provides exemplary residues that correspond to IgV or IgC regions of PD-L2. In some embodiments, the variant PD-L2 polypeptide contains an IgV domain or an IgC domain or specific binding fragments thereof in which the at least one of the amino acid modifications (e.g. substitutions) is in the IgV domain or IgC domain or a specific binding fragment thereof. In some embodiments, the variant PD-L2 polypeptide contains an IgV domain or specific binding fragments thereof in which the at least one of the amino acid modifications (e.g. substitutions) is in the IgV domain or a specific binding fragment thereof. In some embodiments, by virtue of the altered binding activity or affinity, the altered IgV domain or IgC (e.g. IgC2) domain is an affinity-modified IgSF domain.

In some embodiments, the variant is modified in one more IgSF domains relative to the sequence of an unmodified PD-L2 sequence. In some embodiments, the unmodified PD-L2 sequence is a wild-type PD-L2. In some embodiments, the unmodified or wild-type PD-L2 has the sequence of a native PD-L2 or an ortholog thereof. In some embodiments, the unmodified PD-L2 is or comprises the extracellular domain (ECD) of PD-L2 or a portion thereof containing one or more IgSF domain (see Table 2). In some embodiments, the extracellular domain of an unmodified or wild-type PD-L2 polypeptide comprises an IgV domain and an IgC (e.g. IgC2) domain or domains. However, the variant PD-L2 polypeptide need not comprise both the IgV domain and the IgC (e.g. IgC2) domain or domains. In some embodiments, the variant PD-L2 polypeptide comprises or consists essentially of the IgV domain or a specific binding fragment thereof. In some embodiments, the variant PD-L2 polypeptide comprises or consists essentially of one or both of the IgC (e.g. IgC2) domain or specific binding fragments thereof. In some embodiments, the variant PD-L2 polypeptide comprises or consists essentially of only one of the IgC (e.g. IgC2) domain or a specific binding fragment thereof. In some embodiments, the variant PD-L2 polypeptide comprises the IgV domain or a specific binding fragment thereof, and the first and second IgC (e.g. IgC2) domains or specific binding fragment thereof. In some embodiments, the variant PD-L2 is soluble and lacks a transmembrane domain. In some embodiments, the variant PD-L2 further comprises a transmembrane domain and, in some cases, also a cytoplasmic domain.

In some embodiments, the wild-type or unmodified PD-L2 sequence is a mammalian PD-L2 sequence. In some embodiments, the wild-type or unmodified PD-L2 sequence can be a mammalian PD-L2 that includes, but is not limited to, human, mouse, cynomolgus monkey, or rat. In some embodiments, the wild-type or unmodified PD-L2 sequence is human.

In some embodiments, the wild-type or unmodified PD-L2 sequence has (i) the sequence of amino acids set forth in SEQ ID NO:3 or a mature form thereof lacking the signal sequence, (ii) a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:3 or the mature form thereof, or (iii) is a portion of (i) or (ii) containing an IgV domain or IgC (e.g. IgC2) domain or specific binding fragments thereof.

In some embodiments, the wild-type or unmodified PD-L2 sequence is or comprises an extracellular domain of the PD-L2 or a portion thereof. In some embodiments, the unmodified or wild-type PD-L2 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 31, or an ortholog thereof. In some cases, the unmodified or wild-type PD-L2 polypeptide can comprise (i) the sequence of amino acids set forth in SEQ ID NO: 31, (ii) a sequence of amino acids that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 31, or (iii) is a specific binding fragment of the sequence of (i) or (ii) comprising an IgV domain or an IgC (e.g. IgC2) domain.

In some embodiments, the wild-type or unmodified PD-L2 polypeptide comprises an IgV domain or an IgC (e.g. IgC2) domain or domains, or a specific binding fragment thereof. In some embodiments, the IgV domain of the wild-type or unmodified PD-L2 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 55 (corresponding to amino acid residues 21-118 of SEQ ID NO: 4), or an ortholog thereof. For example, the IgV domain of the unmodified or wild-type PD-L2 polypeptide can contain (i) the sequence of amino acids set forth in SEQ ID NO: 55, (ii) a sequence of amino acids that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 55, or (iii) a specific binding fragment of the sequence of (i) or (ii). In some embodiments, the wild-type or unmodified IgV domain is capable of binding one or more PD-L2 cognate binding proteins, such as one or more of PD-1 or RGMb.

In some embodiments, a first IgC2 domain of the wild-type or unmodified PD-L2 polypeptide comprises the amino acid sequence set forth as residues 122-203 of SEQ ID NO: 4, or an ortholog thereof. For example, an IgC2 domain of the unmodified or wild-type PD-L2 polypeptide can contain (i) the sequence of amino acids set forth residues 122-203 of SEQ ID NO: 4, (ii) a sequence of amino acids that has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to residues 122-203 of SEQ ID NO: 4, or (iii) a specific binding fragment of (i) or (ii). In some embodiments, the wild-type or unmodified IgC domain is capable of binding one or more PD-L2 cognate binding proteins.

In some embodiments, the wild-type or unmodified PD-L2 polypeptide contains a specific binding fragment of PD-L2, such as a specific binding fragment of the IgV domain or the IgC (e.g. IgC2) domain. In some embodiments the specific binding fragment can bind PD-1 and/or RGMb. The specific binding fragment can have an amino acid length of at least 50 amino acids, such as at least 60, 70, 80, 90, 100, or 110 amino acids. In some embodiments, a specific binding fragment of the IgV domain contains an amino acid sequence that is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the length of the IgV domain set forth as amino acids 21-118 of SEQ ID NO: 4. In some embodiments, a specific binding fragment of an IgC (e.g. IgC2) domain comprises an amino acid sequence that is at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the length of the IgC domain set forth as amino acids 122-203 of SEQ ID NO: 3.

In some embodiments, the variant PD-L2 polypeptide comprises the ECD domain or a portion thereof comprising one or more affinity modified IgSF domains. In some embodiments, the variant PD-L2 polypeptides can comprise an IgV domain or an IgC (e.g. IgC2) domain or domains, or a specific binding fragment of the IgV domain or a specific binding fragment of the IgC (e.g. IgC2) domain or domains in which one or more of the IgSF domains (IgV or IgC) contains the one or more amino acid modifications (e.g. substitutions). In some embodiments, the variant PD-L2 polypeptides can comprise an IgV domain and an IgC (e.g. IgC2) domain or domains, or a specific binding fragment of the IgV domain and a specific binding fragment of the IgC (e.g. IgC2) domain or domains, in which at least one of the IgV or IgC domain contains the amino acid modifications (e.g. substitutions). In some embodiments, the variant PD-L2 polypeptide comprises a full-length IgV domain. In some embodiments, the variant PD-L2 polypeptide comprises a full-length IgC (e.g. IgC2) domain or domains. In some embodiments, the variant PD-L2 polypeptide comprises a specific binding fragment of the IgV domain. In some embodiments, the variant PD-L2 polypeptide comprises a specific binding fragment of the IgC (e.g. IgC2) domain or domains. In some embodiments, the variant PD-L2 polypeptide comprises a full-length IgV domain and a full-length IgC (e.g. IgC2) domain or domains. In some embodiments, the variant PD-L2 polypeptide comprises a full-length IgV domain and a specific binding fragment of an IgC (e.g. IgC2) domain or domains. In some embodiments, the variant PD-L2 polypeptide comprises a specific binding fragment of an IgV domain and a full-length IgC (e.g. IgC2) domain or domains. In some embodiments, the variant PD-L2 polypeptide comprises a specific binding fragment of an IgV domain and a specific binding fragment of an IgC (e.g. IgC2) domain or domains.

In any of such embodiments, the one or more amino acid modifications (e.g. substitutions) of the variant PD-L2 polypeptides can be located in any one or more of the PD-L2 polypeptide IgSF domains. For example, in some embodiments, one or more amino acid modifications (e.g. substitutions) are located in the extracellular domain of the variant PD-L2 polypeptide. In some embodiments, one or more amino acid modifications (e.g. substitutions) are located in the IgV domain or specific binding fragment of the IgV domain. In some embodiments, one or more amino acid modifications (e.g. substitutions) are located in an IgC (e.g. IgC2) domain or specific binding fragment of an IgC (e.g. IgC2) domain.

Generally, each of the various attributes of polypeptides are separately disclosed below (e.g., soluble and membrane bound polypeptides, affinity of PD-L2 for PD-1 and RGMb, number of variations per polypeptide chain, number of linked polypeptide chains, the number and nature of amino acid alterations per variant PD-L2, etc.). However, as will be clear to the skilled artisan, any particular polypeptide can comprise a combination of these independent attributes. It is understood that reference to amino acids, including to a specific sequence set forth as a SEQ ID NO used to describe domain organization of an IgSF domain are for illustrative purposes and are not meant to limit the scope of the embodiments provided. It is understood that polypeptides and the description of domains thereof are theoretically derived based on homology analysis and alignments with similar molecules. Thus, the exact locus can vary, and is not necessarily the same for each protein. Hence, the specific IgSF domain, such as specific IgV domain or IgC domain, can be several amino acids (such as one, two, three or four) longer or shorter.

Further, various embodiments of the invention as discussed below are frequently provided within the meaning of a defined term as disclosed above. The embodiments described in a particular definition are therefore to be interpreted as being incorporated by reference when the defined term is utilized in discussing the various aspects and attributes described herein. Thus, the headings, the order of presentation of the various aspects and embodiments, and the separate disclosure of each independent attribute is not meant to be a limitation to the scope of the present disclosure.

Exemplary Modifications

Provided herein are variant PD-L2 polypeptides containing at least one affinity-modified IgSF domain (e.g. IgV or IgC) or a specific binding fragment thereof relative to an IgSF domain contained in a wild-type or unmodified PD-L2 polypeptide such that the variant PD-L2 polypeptide exhibits altered (increased or decreased) binding activity or affinity for one or more ligands PD-1 or RGMb compared to a wild-type or unmodified PD-L2 polypeptide. In some embodiments, a variant PD-L2 polypeptide has a binding affinity for PD-1 and/or RGMb that differs from that of a wild-type or unmodified PD-L2 polypeptide control sequence as determined by, for example, solid-phase ELISA immunoassays, flow cytometry, ForteBio Octet or Biacore assays. In some embodiments, the variant PD-L2 polypeptide has an increased binding affinity for PD-1 and/or RGMb. In some embodiments, the variant PD-L2 polypeptide has a decreased binding affinity for PD-1 and/or RGMb, relative to a wild-type or unmodified PD-L2 polypeptide. The PD-1 and/or the RGMb can be a mammalian protein, such as a human protein or a murine protein.

Binding affinities for each of the cognate binding partners are independent; that is, in some embodiments, a variant PD-L2 polypeptide has an increased binding affinity for one or both of PD-1 and/or RGMb, and a decreased binding affinity for one or both of PD-1 and RGMb, relative to a wild-type or unmodified PD-L2 polypeptide.

In some embodiments, the variant PD-L2 polypeptide has an increased binding affinity for PD-1, relative to a wild-type or unmodified PD-L2 polypeptide. In some embodiments, the variant PD-L2 polypeptide has an increased binding affinity for RGMb, relative to a wild-type or unmodified PD-L2 polypeptide. In some embodiments, the variant PD-L2 polypeptide has a decreased binding affinity for PD-1, relative to a wild-type or unmodified PD-L2 polypeptide. In some embodiments, the variant PD-L2 polypeptide has a decreased binding affinity for RGMb, relative to a wild-type or unmodified PD-L2polypeptide.

In some embodiments, the variant PD-L2 polypeptide has an increased binding affinity for PD-1 and RGMb, relative to a wild-type or unmodified PD-L2 polypeptide. In some embodiments, the variant PD-L2 polypeptide has an increased binding affinity for PD-1 and a decreased binding affinity for RGMb, relative to a wild-type or unmodified PD-L2 polypeptide. In some embodiments, the variant PD-L2 polypeptide has a decreased binding affinity for PD-1 and RGMb, relative to a wild-type or unmodified PD-L2 polypeptide. In some embodiments, the variant PD-L2 polypeptide has a decreased binding affinity for PD-1 and an increased binding affinity for RGMb, relative to a wild-type or unmodified PD-L2 polypeptide.

In some embodiments, a variant PD-L2 polypeptide with increased or greater binding affinity to PD-1 and/or RGMb will have an increase in binding affinity relative to the wild-type or unmodified PD-L2 polypeptide control of at least about 5%, such as at least about 10%, 15%, 20%, 25%, 35%, or 50% for the PD-1 and/or RGMb. In some embodiments, the increase in binding affinity relative to the wild-type or unmodified PD-L2 polypeptide is more than 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold 40-fold or 50-fold. In such examples, the wild-type or unmodified PD-L2 polypeptide has the same sequence as the variant PD-L2 polypeptide except that it does not contain the one or more amino acid modifications (e.g. substitutions).

In some embodiments, a variant PD-L2 polypeptide with reduced or decreased binding affinity to PD-1 and/or RGMb will have decrease in binding affinity relative to the wild-type or unmodified PD-L2 polypeptide control of at least 5%, such as at least about 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more for the PD-1 and/or RGMb. In some embodiments, the decrease in binding affinity relative to the wild-type or unmodified PD-L2 polypeptide is more than 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold 40-fold or 50-fold. In such examples, the wild-type or unmodified PD-L2 polypeptide has the same sequence as the variant PD-L2 polypeptide except that it does not contain the one or more amino acid modifications (e.g. substitutions).

In some embodiments, the equilibrium dissociation constant (K_(d)) of any of the foregoing embodiments to PD-1 and/or RGMb can be less than 1×10⁻⁵ M, 1×10⁻⁶ M, 1×10⁻⁷ M, 1×10⁻⁸ M, 1×10⁻⁹ M, 1×10⁻¹⁰ M or 1×10⁻¹¹M, or 1×10⁻¹² M or less.

The wild-type or unmodified PD-L2 sequence does not necessarily have to be used as a starting composition to generate variant PD-L2 polypeptides described herein. Therefore, use of the term “modification”, such as “substitution” does not imply that the present embodiments are limited to a particular method of making variant PD-L2 polypeptides. Variant PD-L2 polypeptides can be made, for example, by de novo peptide synthesis and thus does not necessarily require a modification, such as a “substitution” in the sense of altering a codon to encode for the modification, e.g. substitution. This principle also extends to the terms “addition” and “deletion” of an amino acid residue which likewise do not imply a particular method of making. The means by which the variant PD-L2 polypeptides are designed or created is not limited to any particular method. In some embodiments, however, a wild-type or unmodified PD-L2 encoding nucleic acid is mutagenized from wild-type or unmodified PD-L2 genetic material and screened for desired specific binding affinity and/or induction of IFN-gamma expression or other functional activity. In some embodiments, a variant PD-L2 polypeptide is synthesized de novo utilizing protein or nucleic acid sequences available at any number of publicly available databases and then subsequently screened. The National Center for Biotechnology Information provides such information and its website is publicly accessible via the internet as is the UniProtKB database.

Unless stated otherwise, as indicated throughout the present disclosure, the amino acid modification(s) are designated by amino acid position number corresponding to the numbering of positions of the unmodified ECD sequence set forth in SEQ ID NO:31 or also, where applicable, the unmodified IgV sequence set forth in SEQ ID NO:55 or SEQ ID NO:115 (containing residues 1-102 of SEQ ID NO:31) as follows:

(SEQ ID NO: 31) LFTVTVPKELYIIEHGSNVTLECNFDTGSHVNLGAITASLQKVENDTSPH RERATLLEEQLPLGKASFHIPQVQVRDEGQYQCIIIYGVAWDYKYLTLKV KASYRKINTHILKVPETDEVELTCQATGYPLAEVSWPNVSVPANTSHSRT PEGLYQVTSVLRLKPPPGRNFSCVFWNTHVRELTLASIDLQSQMEPRTHP T (SEQ ID NO: 55) FTVTVPKELYIIEHGSNVTLECNFDTGSHVNLGAITASLQKVENDTSPHR ERATLLEEQLPLGKASFHIPQVQVRDEGQY QCIIIYGVAW DYKYLTLK (SEQ ID NO: 115) LFTVTVPKELYIIEHGSNVTLECNFDTGSHVNLGAITASLQKVENDTSPH RERATLLEEQLPLGKASFHIPQVQVRDEGQY QCIIIYGVAW DYKYLTLKVKA

It is within the level of a skilled artisan to identify the corresponding position of a modification, e.g. amino acid substitution, in a PD-L2 polypeptide, including portion thereof containing an IgSF domain (e.g. IgV) thereof, such as by alignment of a reference sequence with SEQ ID NO:31, SEQ ID NO:55 or SEQ ID NO:115. In the listing of modifications throughout this disclosure, the amino acid position is indicated in the middle, with the corresponding unmodified (e.g. wild-type) amino acid listed before the number and the identified variant amino acid substitution listed after the number. If the modification is a deletion of the position a “del” is indicated and if the modification is an insertion at the position an “ins” is indicated. In some cases, an insertion is listed with the amino acid position indicated in the middle, with the corresponding unmodified (e.g. wild-type) amino acid listed before and after the number and the identified variant amino acid insertion listed after the unmodified (e.g. wild-type) amino acid.

In some embodiments, the variant PD-L2 polypeptide has one or more amino acid modifications, e.g. substitutions in a wild-type or unmodified PD-L2 sequence. The one or more amino acid modifications, e.g. substitutions can be in the ectodomain (extracellular domain) of the wild-type or unmodified PD-L2 sequence. In some embodiments, the one or more amino acid modifications, e.g. substitutions are in the IgV domain or specific binding fragment thereof. In some embodiments, the one or more amino acid modifications, e.g. substitutions are in an IgC (e.g. IgC2) domain or specific binding fragment thereof. In some embodiments of the variant PD-L2 polypeptide, some of the one or more amino acid modifications, e.g. substitutions are in the IgV domain or a specific binding fragment thereof, and some of the one or more amino acid modifications, e.g. substitutions are in an IgC domain or domains (e.g. IgC2) or a specific binding fragment thereof.

In some embodiments, the variant PD-L2 polypeptide has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications, e.g. substitutions. The modifications, e.g. substitutions can be in the IgV domain or the IgC (e.g. IgC2) domain or domains. In some embodiments, the variant PD-L2 polypeptide has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications, e.g. substitutions in the IgV domain or specific binding fragment thereof. In some embodiments, the variant PD-L2 polypeptide has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid modifications, e.g. substitutions in the IgC (e.g. IgC2) domain or domains or specific binding fragment thereof. In some embodiments, the variant PD-L2 polypeptide has at least about 85%, 86%, 86%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the wild-type or unmodified PD-L2 polypeptide or specific binding fragment thereof, such as with the amino acid sequence of SEQ ID NO: 31, 55 or 115.

In some embodiments, the variant PD-L2 polypeptide has one or more amino acid modifications, e.g. substitutions in an unmodified PD-L2 or specific binding fragment thereof corresponding to position(s) 2, 12, 13, 15, 18, 20, 23, 24, 28, 31, 32, 33, 36, 37, 39, 44, 45, 46, 47, 48, 58, 59, 65, 67, 69, 71, 72, 73, 74, 75, 76, 77, 82, 85, 86, 89, or 91 with reference to positions set forth in SEQ ID NO: 31. In some embodiments, such variant PD-L2 polypeptides exhibit altered binding affinity to one or more of PD-1 and/or RGMb compared to the wild-type or unmodified PD-L2 polypeptide. For example, in some embodiments, the variant PD-L2 polypeptide exhibits increased binding affinity to PD-1 and/or RGMb compared to a wild-type or unmodified PD-L2 polypeptide. In some embodiments, the variant PD-L2 polypeptide exhibits decreased binding affinity to PD-1 or RGMb compared to a wild-type or unmodified PD-L2 polypeptide.

In some embodiments, the variant PD-L2 polypeptide has one or more amino acid modifications, e.g. amino acid substitutions, selected from F2L, 112V, I13V, H15Q, N18D, T20A, N24S, C23S, G28V, N24D, V31A, V31M, N32D, L33P, L33H, L33F, I36V, T37A, S48C, S39I, E44D, E44V, N45S, D46E, T47A, E58G, E59G, K65R, S67L, H69L, P71S, Q72H, V73A, Q74R, V75G, R76G, D77N, Q82R, I85F, I86T, V89D, or W91R, or a conservative amino acid substitution thereof. A conservative amino acid substitution is any amino acid that falls in the same class of amino acids as the substituted amino acids, other than the wild-type or unmodified amino acid. The classes of amino acids are aliphatic (glycine, alanine, valine, leucine, and isoleucine), hydroxyl or sulfur-containing (serine, cysteine, threonine, and methionine), cyclic (proline), aromatic (phenylalanine, tyrosine, tryptophan), basic (histidine, lysine, and arginine), and acidic/amide (aspartate, glutamate, asparagine, and glutamine).

In some embodiments, the variant PD-L2 polypeptide has two or more amino acid modifications, e.g. amino acid substitutions, selected from F2L, I12V, I13V, H15Q, N18D, T20A, N24S, C23S, G28V, N24D, V31A, V31M, N32D, L33P, L33H, L33F, I36V, T37A, S48C, S39I, E44D, E44V, N45S, D46E, T47A, E58G, E59G, K65R, S67L, H69L, P71S, Q72H, V73A, Q74R, V75G, R76G, D77N, Q82R, I85F, I86T, V89D, or W91R.

In some embodiments, the amino acid modifications, e.g. amino acid substitutions, of a variant PD-L2 polypeptide include H15Q, N24D, E44D, V89D, Q82R/V89D, E59G/Q82R, S39I/V89D, S67L/V89D, S67L/I85F, S67L/I86T, H15Q/K65R, H15Q/Q72H/V89D, H15Q/S67L/R76G, H15Q/R76G/I85F, H15Q/T47A/Q82R, H15Q/Q82R/V89D, H15Q/C23S/I86T, H15Q/S39I/I86T, E44D/V89D/W91R, I13V/S67L/V89D, H15Q/S67L/I86T, I13V/H15Q/S67L/I86T, I13V/H15Q/E44D/V89D, I13V/S39I/E44D/Q82R/V89D, I13V/E44D/Q82R/V89D, I13V/Q72H/R76G/I86T, I13V/H15Q/R76G/I85F, H15Q/S39I/R76G/V89D, H15Q/S67L/R76G/I85F, H15Q/T47A/Q72H/R76G/I86T, H15Q/T47A/Q72H/R76G, I13V/H15Q/T47A/Q72H/R76G, H15Q/E44D/R76G/I85F, H15Q/S39I/S67L/V89D, H15Q/N32D/S67L/V89D, N32D/S67L/V89D, H15Q/S67L/Q72H/R76G/V89D, H15Q/Q72H/Q74R/R76G/I86T, G28V/Q72H/R76G/I86T, I13V/H15Q/S39I/E44D/S67L, E44D/S67L/Q72H/Q82R/V89D, H15Q/V89D, H15Q/T47A, I13V/H15Q/Q82R, I13V/H15Q/V89D, I13V/S67L/Q82R/V89D, I13V/H15Q/Q82R/V89D, H15Q/V31M/S67L/Q82R/V89D, I13V/H15Q/T47A/Q82R, I13V/H15Q/V31A/N45S/Q82R/V89D, H15Q/T47A/H69L/Q82R/V89D, I13V/H15Q/T47A/H69L/R76G/V89D, I12V/I13V/H15Q/T47A/Q82R/V89D, I13V/H15Q/R76G/D77N/Q82R/V89D, I13V/H15Q/T47A/R76G/V89D, I13V/H15Q/T47A/Q82R/V89D, I13V/H15Q/N24D/Q82R/V89D, I13V/H15Q/I36V/T47A/S67L/V89D, H15Q/T47A/K65R/S67L/Q82R/V89D, H15Q/L33P/T47A/S67L/P71S/V89D, I13V/H15Q/Q72H/R76G/I86T, H15Q/T47A/S67L/Q82R/V89D, F2L/H15Q/D46E/T47A/Q72H/R76G/Q82R/V89D, I13V/H15Q/L33F/T47A/Q82R/V89D, I13V/H15Q/T47A/E58G/S67L/Q82R/V89D, H15Q/N24S/T47A/Q72H/R76G/V89D, I13V/H15Q/E44V/T47A/Q82R/V89D, H15Q/N18D/T47A/Q72H/V73A/R76G/I86T/V89D, I13V/H15Q/T37A/E44D/S48C/S67L/Q82R/V89D, H15Q/L33H/S67L/R76G/Q82R/V89D, I13V/H15Q/T47A/Q72H/R76G/I86T, H15Q/S39I/E44D/Q72H/V75G/R76G/Q82R/V89D, H15Q/T47A/S67L/R76G/Q82R/V89D, or I13V/H15Q/T47A/S67L/Q72H/R76G/Q82R/V89D.

In some embodiments, the variant PD-L2 polypeptide comprises amino acid modifications in an unmodified PD-L2 or specific binding fragment thereof at a position corresponding to position 13, with reference to positions set forth in SEQ ID NO: 31. In some embodiments, the amino acid modification is the amino acid substitution I13V or a conservative amino acid substitution thereof. In some embodiments, the variant PD-L2 polypeptide further contains one or more amino acid modifications, e.g. amino acid substitutions, at one or more positions 15, 44, 47, 67, 72, 72, 76, 82, 86 or 89. In some embodiments, the one or more amino acid modification is one or more amino acid substitutions H15Q, E44D, T47A, S67L, Q72H, R76G, Q82R, I86T, V89D, or a conservative amino acid substitution thereof. In some embodiments, the variant PD-L2 polypeptide comprises the amino acid modifications I13V/H15Q, I13V/E44D, I13V/T47A, I13V/S67L, I13V/Q72H, I13V/R76G, I13V/Q82R, I13V/I86T or I13V/V89D.

In some embodiments, the variant PD-L2 polypeptide comprises amino acid modifications in an unmodified PD-L2 or specific binding fragment thereof at a position corresponding to position 15, with reference to positions set forth in SEQ ID NO: 31. In some embodiments, the amino acid modification is the amino acid substitution H15Q or a conservative amino acid substitution thereof. In some embodiments, the variant PD-L2 polypeptide further contains one or more amino acid modifications, e.g. amino acid substitutions, at one or more positions 13, 44, 47, 67, 72, 72, 76, 82, 86 or 89. In some embodiments, the one or more amino acid modification is one or more amino acid substitutions I13V, E44D, T47A, S67L, Q72H, R76G, Q82R, I86T, V89D, or a conservative amino acid substitution thereof. In some embodiments, the variant PD-L2 polypeptide comprises the amino acid modifications I13V/H15Q, H15Q/E44D, H15Q/T47A, H15Q/S67L, H15Q/Q72H, H15Q/R76G, H15Q/Q82R, H15Q/I86T or H15Q/V89D. In some embodiments, the variant PD-L2 polypeptide comprises the amino acid modifications H15Q/S62L/Q82R, H15Q/S62L/V89D, or H15Q/Q82R/V89D.

In some embodiments, the variant PD-L2 polypeptide comprises amino acid modifications in an unmodified PD-L2 or specific binding fragment thereof at a position corresponding to position 47, with reference to positions set forth in SEQ ID NO: 31. In some embodiments, the amino acid modification is the amino acid substitution T47A or a conservative amino acid substitution thereof. In some embodiments, the variant PD-L2 polypeptide further contains one or more amino acid modifications, e.g. amino acid substitutions, at one or more positions 13, 15, 44, 67, 72, 72, 76, 82, 86 or 89. In some embodiments, the one or more amino acid modification is one or more amino acid substitutions I13V, H15Q, E44D, S67L, Q72H, R76G, Q82R, I86T, V89D, or a conservative amino acid substitution thereof. In some embodiments, the variant PD-L2 polypeptide comprises the amino acid modifications I13V/T47A, H15Q/T47A, T47A/E44D, T47A/S67L, T47A/Q72H, T47A/R76G, T47A/Q82R, T47A/I86T or T47A/V89D.

In some embodiments, the variant PD-L2 polypeptide comprises amino acid modifications in an unmodified PD-L2 or specific binding fragment thereof at a position corresponding to position 67, with reference to positions set forth in SEQ ID NO: 31. In some embodiments, the amino acid modification is the amino acid substitution S67L or a conservative amino acid substitution thereof. In some embodiments, the variant PD-L2 polypeptide further contains one or more amino acid modifications, e.g. amino acid substitutions, at one or more positions 13, 15, 44, 47, 72, 72, 76, 82, 86 or 89. In some embodiments, the one or more amino acid modification is one or more amino acid substitutions I13V, H15Q, E44D, T47A, Q72H, R76G, Q82R, I86T, V89D, or a conservative amino acid substitution thereof. In some embodiments, the variant PD-L2 polypeptide comprises the amino acid modifications I13V/S67L, H15Q/S67L, S67L/E44D, T47A/S67L, S67L/Q72H, S67L/R76G, S67L/Q82R, S67L/I86T or S67L/V89D. In some embodiments, the variant PD-L2 polypeptide comprises the amino acid modifications H15Q/S62L/Q82R, H15Q/S62L/V89D, or S62L/Q82R/V89D.

In some embodiments, the variant PD-L2 polypeptide comprises amino acid modifications in an unmodified PD-L2 or specific binding fragment thereof at a position corresponding to position 72, with reference to positions set forth in SEQ ID NO: 31. In some embodiments, the amino acid modification is the amino acid substitution Q72H or a conservative amino acid substitution thereof. In some embodiments, the variant PD-L2 polypeptide further contains one or more amino acid modifications, e.g. amino acid substitutions, at one or more positions 13, 15, 44, 47, 67, 76, 82, 86 or 89. In some embodiments, the one or more amino acid modification is one or more amino acid substitutions I13V, H15Q, E44D, T47A, S67L, R76G, Q82R, I86T, V89D, or a conservative amino acid substitution thereof. In some embodiments, the variant PD-L2 polypeptide comprises the amino acid modifications I13V/Q72H, H15Q/Q72H, Q72H/E44D, T47A/Q72H, S67L/Q72H, Q72H/R76G, Q72H/Q82R, Q72H/I86T or Q72H/V89D.

In some embodiments, the variant PD-L2 polypeptide comprises amino acid modifications in an unmodified PD-L2 or specific binding fragment thereof at a position corresponding to position 76, with reference to positions set forth in SEQ ID NO: 31. In some embodiments, the amino acid modification is the amino acid substitution R76G or a conservative amino acid substitution thereof. In some embodiments, the variant PD-L2 polypeptide further contains one or more amino acid modifications, e.g. amino acid substitutions, at one or more positions 13, 15, 44, 47, 67, 72, 82, 86 or 89. In some embodiments, the one or more amino acid modification is one or more amino acid substitutions I13V, H15Q, E44D, T47A, S67L, Q72H, Q82R, I86T, V89D, or a conservative amino acid substitution thereof. In some embodiments, the variant PD-L2 polypeptide comprises the amino acid modifications I13V/R76G, H15Q/R76G, E44D/R76G, T47A/R76G, S67L/R76G, Q72H/R76G, R76G/Q82R, R76G/I86T or R76G/V89D.

In some embodiments, the variant PD-L2 polypeptide comprises amino acid modifications in an unmodified PD-L2 or specific binding fragment thereof at a position corresponding to position 82, with reference to positions set forth in SEQ ID NO: 31. In some embodiments, the amino acid modification is the amino acid substitution Q82R or a conservative amino acid substitution thereof. In some embodiments, the variant PD-L2 polypeptide further contains one or more amino acid modifications, e.g. amino acid substitutions, at one or more positions 13, 15, 44, 47, 67, 72, 76, 86 or 89. In some embodiments, the one or more amino acid modification is one or more amino acid substitutions I13V, H15Q, E44D, T47A, S67L, Q72H, R76G, I86T, V89D, or a conservative amino acid substitution thereof. In some embodiments, the variant PD-L2 polypeptide comprises the amino acid modifications I13V/Q82R, H15Q/Q82R, E44D/Q82R, T47A/Q82R, S67L/Q82A, Q72H/Q82R, R76G/Q82R, Q82R/I86T or Q86R/V89D.

In some embodiments, the variant PD-L2 polypeptide comprises amino acid modifications in an unmodified PD-L2 or specific binding fragment thereof at a position corresponding to position 86, with reference to positions set forth in SEQ ID NO: 31. In some embodiments, the amino acid modification is the amino acid substitution I86T or a conservative amino acid substitution thereof. In some embodiments, the variant PD-L2 polypeptide further contains one or more amino acid modifications, e.g. amino acid substitutions, at one or more positions 13, 15, 44, 47, 67, 72, 76, 82 or 89. In some embodiments, the one or more amino acid modification is one or more amino acid substitutions I13V, H15Q, E44D, T47A, S67L, Q72H, R76G, Q82R, V89D, or a conservative amino acid substitution thereof. In some embodiments, the variant PD-L2 polypeptide comprises the amino acid modifications I13V/I86T, H15Q/I86T, E44D/I86T, T47A/I86T, S67L/I86T, Q72H/I86T, R76G/I86T, Q82R/I86T or I86T/V89D.

In some embodiments, the variant PD-L2 polypeptide comprises amino acid modifications in an unmodified PD-L2 or specific binding fragment thereof at a position corresponding to position 89, with reference to positions set forth in SEQ ID NO: 31. In some embodiments, the amino acid modification is the amino acid substitution V89D or a conservative amino acid substitution thereof. In some embodiments, the variant PD-L2 polypeptide further contains one or more amino acid modifications, e.g. amino acid substitutions, at one or more positions 13, 15, 44, 47, 67, 72, 76, 82 or 86. In some embodiments, the one or more amino acid modification is one or more amino acid substitutions I13V, H15Q, E44D, T47A, S67L, Q72H, R76G, Q82R, I86T, or a conservative amino acid substitution thereof. In some embodiments, the variant PD-L2 polypeptide comprises the amino acid modifications I13V/V89D, H15Q/V89D, E44D/V89D, T47A/V89D, S67L/V89D, Q72H/V89D, R76H/V89D, I86T/V89D. In some embodiments, the variant PD-L2 polypeptide comprises the amino acid modifications H15Q/S62L/V89D, H15Q/Q82R/V89D, or S62L/Q82R/V89D.

In some embodiments, the variant PD-L2 polypeptide comprises any of the substitutions (mutations) listed in Table 1. Table 1 also provides exemplary sequences by reference to SEQ ID NO for the extracellular domain (ECD) or IgV domain of wild-type PD-L2 or exemplary variant PD-L2 polypeptides. As indicated, the exact locus or residues corresponding to a given domain can vary, such as depending on the methods used to identify or classify the domain. Also, in some cases, adjacent N- and/or C-terminal amino acids of a given domain (e.g. IgV) also can be included in a sequence of a variant IgSF polypeptide, such as to ensure proper folding of the domain when expressed. Thus, it is understood that the exemplification of the SEQ ID NOS in Table 1 is not to be construed as limiting. For example, the particular domain, such as the IgV domain, of a variant PD-L2 polypeptide can be several amino acids longer or shorter, such as 1-10, e.g. 1, 2, 3, 4, 5, 6 or 7 amino acids longer or shorter, than the sequence of amino acids set forth in the respective SEQ ID NO.

In some embodiments, the variant PD-L2 polypeptide comprises any of the mutations listed in Table 1. Table 1 also provides exemplary sequences by reference to SEQ ID NO for the extracellular domain (ECD) or IgV domain of wild-type PD-L2 or exemplary variant PD-L2 polypeptides. As indicated, the exact locus or residues corresponding to a given domain can vary, such as depending on the methods used to identify or classify the domain. Also, in some cases, adjacent N- and/or C-terminal amino acids of a given domain (e.g. IgV) also can be included in a sequence of a variant IgSF polypeptide, such as to ensure proper folding of the domain when expressed. Thus, it is understood that the exemplification of the SEQ ID NOs in Table 1 is not to be construed as limiting. For example, the particular domain, such as the IgV domain, of a variant PD-L2 polypeptide can be several amino acids longer or shorter, such as 1-10, e.g. 1, 2, 3, 4, 5, 6 or 7 amino acids longer or shorter, than the sequence of amino acids set forth in the respective SEQ ID NO.

In some embodiments, the variant PD-L2 polypeptide comprises any of the mutations listed in Table 1.

In some embodiments, the variant PD-L2 polypeptide comprises or consists of any of the extracellular domain (ECD) sequences listed in Table 1 (i.e., any one of SEQ ID NOS: 56-106, 108-114 and 116-132). In some embodiments, the variant PD-L2 polypeptide comprises or consists of a polypeptide sequence that exhibits at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, such as at least 96% identity, 97% identity, 98% identity, or 99% identity to any of the extracellular domain (ECD) sequences listed in Table 1 (i.e., any one of SEQ ID NOS: 56-106, 108-114 and 116-132) and contains the amino acid modification(s), e.g. substitution(s) not present in the wild-type or unmodified PD-L2. In some embodiments, the variant PD-L2 polypeptide comprises or consists of a specific binding fragment of any of the extracellular domain (ECD) sequences listed in Table 1 (i.e., any one of SEQ ID NOS: 56-106, 108-114 and 116-132) and contains the amino acid modification(s), e.g. substitution(s) not present in the wild-type or unmodified PD-L2.

In some embodiments, the variant PD-L2 polypeptide comprises or consists of any of the IgV sequences listed in Table 1 (i.e., any one of SEQ ID NOS: 133-183, 185-191, 193-209, 268-318, 320-343). In some embodiments, the variant PD-L2 polypeptide comprises or consists of a polypeptide sequence that exhibits at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, such as at least 96% identity, 97% identity, 98% identity, or 99% identity to any of the IgV sequences listed in Table 1 (i.e., any one of SEQ ID NOS: 133-183, 185-191, 193-209, 268-318, 320-343) and contains the amino acid substitution(s) not present in the wild-type or unmodified PD-L2. In some embodiments, the variant PD-L2 polypeptide comprises or consists of a specific binding fragment of any of the IgV sequences listed in Table 1 (i.e., any one of SEQ ID NOS: 133-183, 185-191, 193-209, 268-318, 320-343) and contains the amino acid modification(s), e.g. substitution(s) not present in the wild-type or unmodified PD-L2.

TABLE 1 Exemplary variant PD-L2 polypeptides ECD SEQ ID IgV SEQ ID Mutation(s) NO NO Wild-type 31 55, 115 H15Q 56 133, 268 N24D 57 134, 269 E44D 58 135, 270 V89D 59 136, 271 Q82R/V89D 60 137, 272 E59G/Q82R 61 138, 273 S39I/V89D 62 139, 274 S67L/V89D 63 140, 275 S67L/I85F 64 141, 276 S67L/I86T 65 142, 277 H15Q/K65R 66 143, 278 H15Q/Q72H/V89D 67 144, 279 H15Q/S67L/R76G 68 145, 280 H15Q/R76G/I85F 69, 74 146, 281, 151, 186 H15Q/T47A/Q82R 70 147, 282 H15Q/Q82R/V89D 71 148, 283 H15Q/C23S/I86T 72 149, 284 H15Q/S39I/I86T 73 150, 285 E44D/V89D/W91R 75 152, 287 I13V/S67L/V89D 76 153, 288 H15Q/S67L/I86T 77 154, 289 I13V/H15Q/S67L/I86T 78 155, 290 I13V/H15Q/E44D/V89D 79 156, 291 I13V/S39I/E44D/Q82R/V89D 80 157, 292 I13V/E44D/Q82R/V89D 81 158, 293 I13V/Q72H/R76G/I86T 82 159, 294 I13V/H15Q/R76G/I85F 83 160, 295 H15Q/S39I/R76G/V89D 84 161, 296 H15Q/S67L/R76G/I85F 85 162, 297 H15Q/T47A/Q72H/R76G/I86T 86 163, 298 H15Q/T47A/Q72H/R76G 87 164, 299 I13V/H15Q/T47A/Q72H/R76G 88 165, 300 H15Q/E44D/R76G/I85F 89 166, 301 H15Q/S39I/S67L/V89D 90 167, 302 H15Q/N32D/S67L/V89D 91 168, 303 N32D/S67L/V89D 92 169, 304 H15Q/S67L/Q72H/R76G/V89D 93 170, 305 H15Q/Q72H/Q74R/R76G/I86T 94 171, 306 G28V/Q72H/R76G/I86T 95 172, 307 I13V/H15Q/S39I/E44D/S67L 96 173, 308 E44D/S67L/Q72H/Q82R/V89D 97 174, 309 H15Q/V89D 98 175, 310 H15Q/T47A 99 176, 311 I13V/H15Q/Q82R 100 177, 312 I13V/H15Q/V89D 101 178, 313 I13V/S67L/Q82R/V89D 102 179, 314 I13V/H15Q/Q82R/V89D 103 180, 315 H15Q/V31M/S67L/Q82R/V89D 104 181, 316 I13V/H15Q/T47A/Q82R 105 182, 317 I13V/H15Q/V31A/N45S/Q82R/V89D 106 183, 318 H15Q/T47A/H69L/Q82R/V89D 108 185, 320 I13V/H15Q/T47A/H69L/R76G/V89D 109 186, 321 I12V/I13V/H15Q/T47A/Q82R/V89D 110 187, 322 I13V/H15Q/R76G/D77N/Q82R/V89D 111 188, 323 I13V/H15Q/T47A/R76G/V89D 112 189, 324 I13V/H15Q/T47A/Q82R/V89D 113 190, 325 I13V/H15Q/N24D/Q82R/V89D 114 191, 326 I13V/H15Q/I36V/T47A/S67L/V89D 116 193, 327 H15Q/T47A/K65R/S67L/Q82R/V89D 117 194, 328 H15Q/L33P/T47A/S67L/P71S/V89D 118 195, 329 I13V/H15Q/Q72H/R76G/I86T 119 196, 330 H15Q/T47A/S67L/Q82R/V89D 120 197, 331 F2L/H15Q/D46E/T47A/Q72H/R76G/ 121 198, 332 Q82R/V89D I13V/H15Q/L33F/T47A/Q82R/V89D 122 199, 333 I13V/H15Q/T47A/E58G/S67L/Q82R/V89D 123 200, 334 H15Q/N24S/T47A/Q72H/R76G/V89D 124 201, 335 I13V/H15Q/E44V/T47A/Q82R/V89D 125 202, 336 H15Q/N18D/T47A/Q72H/V73A/R76G/ 126 203, 337 I86T/V89D I13V/H15Q/T37A/E44D/S48C/S67L/ 127 204, 338 Q82R/V89D H15Q/L33H/S67L/R76G/Q82R/V89D 128 205, 339 I13V/H15Q/T47A/Q72H/R76G/I86T 129 206, 340 H15Q/S39I/E44D/Q72H/V75G/R76G/ 130 207, 341 Q82R/V89D H15Q/T47A/S67L/R76G/Q82R/V89D 131 208, 342 I13V/H15Q/T47A/S67L/Q72H/R76G/ 132 209, 343 Q82R/V89D

In some embodiments, the variant PD-L2 polypeptide exhibits increased affinity for the ectodomain of PD-1 compared to the wild-type or unmodified PD-L2 polypeptide, such as comprising the sequence set forth in SEQ ID NO: 31, 55 or 115. In some embodiments, the PD-L2 polypeptide exhibits increased affinity for the ectodomain of RGMb compared to the wild-type or unmodified PD-L2, such as comprising the sequence set forth in SEQ ID NO: 31, 55 or 115. In some embodiments, the PD-L2 polypeptide exhibits increased affinity for the ectodomain of PD-1 and the ectodomain of RGMb compared to the wild-type or unmodified PD-L2, such as comprising the sequence set forth in SEQ ID NO: 31, 55 or 115.

In some embodiments, the variant PD-L2 polypeptide exhibits increased binding affinity for binding one of the ectodomains of PD-1 or RGMb and exhibits decreased binding affinity for binding to the other of the ectodomains of PD-1 or RGMb compared to the wild-type or unmodified PD-L2 polypeptide, such as comprising the sequence set forth in SEQ ID NO: 31, 55 or 115. In some embodiments, the variant PD-L2 polypeptide exhibits increased affinity for the ectodomain of PD-1, and decreased affinity for the ectodomain of RGMb, compared to wild-type or unmodified PD-L2 polypeptide, such as comprising the sequence set forth in SEQ ID NO: 31, 55 or 115. In some embodiments, the variant PD-L2 polypeptide exhibits increased affinity for the ectodomain of RGMb and decreased affinity for the ectodomain of PD-1, compared to wild-type or unmodified PD-L2 polypeptide, such as comprising the sequence set forth in SEQ ID NO: 31, 55 or 115.

In some embodiments, a variant PD-L2 polypeptide exhibits increased selectivity for PD-1 versus RGMb or other cognate binding partner of PD-L2 compared to the ratio of binding of the unmodified PD-L2 polypeptide, e.g. set forth in SEQ ID NO: 31, 55 or 115, for binding RGMb or other cognate binding partner. In some embodiments, the ratio of PD-1 binding to RGMb binding (PD-1:RGMb binding ratio) is greater than 1. In some embodiments, the variant PD-L2 polypeptide exhibits a ratio of binding PD-1 versus RGMb or other cognate binding partner that is greater than or greater than about or 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, or more.

III. FORMAT OF VARIANT POLYPEPTIDES

The immunomodulatory polypeptide comprising a variant PD-L2 provided herein in which is contained a vIgD can be formatted in a variety of ways, including as a soluble protein, membrane bound protein or secreted protein. In some embodiments, the particular format can be chosen for the desired therapeutic application. In some cases, an immunomodulatory polypeptide comprising a variant PD-L2 polypeptide is provided in a format to antagonize or block activity of its cognate binding partner, e.g. PD-1. In some embodiments, antagonism of PD-1 may be useful to promote immunity in oncology. In some cases, an immunomodulatory polypeptide comprising a variant PD-L2 polypeptide is provided in a format to agonize or stimulate activity of its cognate binding partner, e.g. PD-1. In some embodiments, agonism of PD-1 may be useful for treating inflammation or autoimmunity. A skilled artisan can readily determine the activity of a particular format, such as for antagonizing or agonizing one or more specific cognate binding partner. Exemplary methods for assessing such activities are provided herein, including in the examples.

In some aspects, provided are immunomodulatory proteins comprising a vIgD of PD-L2 in which such proteins are soluble, e.g. fused to an Fc chain. In some aspects, one or more additional IgSF domain, such as one or more additional vIgD, may be linked to a vIgD of PD-L2 as provided herein (hereinafter called a “stack” or “stacked” immunomodulatory protein). In some embodiments, the modular format of the provided immunomodulatory proteins provides flexibility for engineering or generating immunomodulatory proteins for modulating activity of multiple counter structures (multiple cognate binding partners). In some embodiments, such “stack” molecules can be provided in a soluble format or, in some cases, may be provided as membrane bound or secreted proteins. In some embodiments, a variant PD-L2 immunomodulatory protein is provided as a conjugate in which is contained a vIgD of PD-L2 linked, directly or indirectly, to a targeting agent or moiety, e.g. to an antibody or other binding molecules that specifically binds to a ligand, e.g. an antigen, for example, for targeting or localizing the vIgD to a specific environment or cell, such as when administered to a subject. In some embodiments, the targeting agent, e.g. antibody or other binding molecule, binds to a tumor antigen, thereby localizing the variant PD-L2 containing the vIgD to the tumor microenvironment, for example, to modulate activity of tumor infiltrating lymphocytes (TILs) specific to the tumor microenvironment.

In some embodiments, provided immunomodulatory proteins are expressed in cells and provided as part of an engineered cellular therapy (ECT). In some embodiments, the variant PD-L2 polypeptide is expressed in a cell, such as an immune cell (e.g. T cell or antigen presenting cell), in membrane-bound form, thereby providing a transmembrane immunomodulatory protein (hereinafter also called a “TIP”). In some embodiments, depending on the cognate binding partner recognized by the TIP, engineered cells expressing a TIP can agonize a cognate binding partner by providing a costimulatory signal, either positive to negative, to other engineered cells and/or to endogenous T cells. In some aspects, the variant PD-L2 polypeptide is expressed in a cell, such as an immune cell (e.g. T cell or antigen presenting cell), in secretable form to thereby produce a secreted or soluble form of the variant PD-L2 polypeptide (hereinafter also called a “SIP”), such as when the cells are administered to a subject. In some aspects, a SIP can antagonize a cognate binding partner in the environment (e.g. tumor microenvironment) in which it is secreted. In some embodiments, a variant PD-L2 polypeptide is expressed in an infectious agent (e.g. viral or bacterial agent) which, upon administration to a subject, is able to infect a cell in vivo, such as an immune cell (e.g. T cell or antigen presenting cell), for delivery or expression of the variant polypeptide as a TIP or a SIP in the cell.

In some embodiments, a soluble immunomodulatory polypeptide, such as a variant PD-L2 containing a vIgD, can be encapsulated within a liposome which itself can be conjugated to any one of or any combination of the provided conjugates (e.g., a targeting moiety). In some embodiments, the soluble or membrane bound immunomodulatory polypeptides of the invention are deglycosylated. In more specific embodiments, the variant PD-L2 sequence is deglycosylated. In even more specific embodiments, the IgV and/or IgC (e.g. IgC2) domain or domains of the variant PD-L2 is deglycosylated.

Non-limiting examples of provided formats are described in FIGS. 1A-1C and further described below.

A. Soluble Protein

In some embodiments, the immunomodulatory protein containing a variant PD-L2 polypeptide is a soluble protein. Those of skill will appreciate that cell surface proteins typically have an intracellular, transmembrane, and extracellular domain (ECD) and that a soluble form of such proteins can be made using the extracellular domain or an immunologically active subsequence thereof. Thus, in some embodiments, the immunomodulatory protein containing a variant PD-L2 polypeptide lacks a transmembrane domain or a portion of the transmembrane domain. In some embodiments, the immunomodulatory protein containing a variant PD-L2 lacks the intracellular (cytoplasmic) domain or a portion of the intracellular domain. In some embodiments, the immunomodulatory protein containing the variant PD-L2 polypeptide only contains the vIgD portion containing the ECD domain or a portion thereof containing an IgV domain and/or IgC (e.g. IgC2) domain or domains or specific binding fragments thereof containing the amino acid modification(s).

In some embodiments, the immunomodulatory protein is or contains a variant PD-L2 polypeptide that is in monomer form and/or that exhibits monovalent binding to its binding partner. In some aspects, a variant PD-L2 polypeptide as described, such as a variant PD-L2 that is soluble and/or that lacks a transmembrane domain and intracellular signaling domain, is linked, directly or indirectly, to a further moiety. In some embodiments, the further moiety is a protein, peptide, small molecule or nucleic acid. In some embodiments, the monovalent immunomodulatory protein is a fusion protein. In some embodiments, the moiety is a half-life extending molecule. Exemplary of such half-life extending molecules include, but are not limited to, albumin, an albumin-binding polypeptide, Pro/Ala/Ser (PAS), a C-terminal peptide (CTP) of the beta subunit of human chorionic gonadotropin, polyethylene glycol (PEG), long unstructured hydrophilic sequences of amino acids (XTEN), hydroxyethyl starch (HES), an albumin-binding small molecule, or a combination thereof.

In some embodiments, the immunomodulatory polypeptide comprising a variant PD-L2 can be linked to a moiety that includes conformationally disordered polypeptide sequences composed of the amino acids Pro, Ala, and Ser (See e.g., WO2008/155134, SEQ ID NO: 2033). In some cases, the amino acid repeat is at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acid residues, wherein each repeat comprises (an) Ala, Ser, and Pro residue(s). Thus, provided herein is an immunomodulatory protein is a PASylated protein wherein the variant PD-L1 polypeptide is linked, directly or indirectly via a linker, to Pro/Ala/Ser (PAS). In some embodiments, one or more additional linker structures may be used.

In some embodiments, the moiety facilitates detection or purification of the variant PD-L2 polypeptide. In some cases, the immunomodulatory polypeptide comprises a tag or fusion domain, e.g. affinity or purification tag, linked, directly or indirectly, to the N- and/or c-terminus of the PD-L2 polypeptide. Various suitable polypeptide tags and/or fusion domains are known, and include but are not limited to, a poly-histidine (His) tag, a FLAG-tag (SEQ ID NO: 2034), a Myc-tag, and fluorescent protein-tags (e.g., EGFP, set forth in SEQ ID NOs: 2035-2037). In some cases, the immunomodulatory polypeptide comprising a variant PD-L2 comprises at least six histidine residues (set forth in SEQ ID NO: 1253). In some cases, the immunomodulatory polypeptide comprising a variant PD-L2 further comprises various combinations of moieties. For example, the immunomodulatory polypeptide comprising a variant PD-L2 further comprises one or more polyhistidine-tag and FLAG tag.

In some embodiments, the PD-L2 polypeptide is linked to a modified immunoglobulin heavy chain constant region (Fc) that remains in monovalent form such as set forth in SEQ ID NO: 1187.

In some embodiments, the immunomodulatory protein contains a variant PD-L2 polypeptide that is linked, directly or indirectly via a linker to a multimerization domain. In some aspects, the multimerization domain increases half-life of the molecule. Interaction of two or more variant PD-L2 polypeptides can be facilitated by their linkage, either directly or indirectly, to any moiety or other polypeptide that are themselves able to interact to form a stable structure. For example, separate encoded variant PD-L2 polypeptide chains can be joined by multimerization, whereby multimerization of the polypeptides is mediated by a multimerization domain. Typically, the multimerization domain provides for the formation of a stable protein-protein interaction between a first variant PD-L2 polypeptide and a second variant PD-L2 polypeptide.

Homo- or heteromultimeric polypeptides can be generated from co-expression of separate variant PD-L2 polypeptides. The first and second variant PD-L2 polypeptides can be the same or different. In particular embodiments, the first and second variant PD-L2 polypeptide are the same in a homodimer, and each are linked to a multimerization domain that is the same. In other embodiments, heterodimers can be formed by linking first and second variant PD-L2 polypeptides that are different. In such embodiments, in some aspects the first and second variant PD-L2 polypeptide are linked to different multimerization domains capable of promoting heterodimer formation.

In some embodiments, a multimerization domain includes any capable of forming a stable protein-protein interaction. The multimerization domains can interact via an immunoglobulin sequence (e.g. Fc domain; see e.g., International Patent Pub. Nos. WO 93/10151 and WO 2005/063816 US; U.S. Pub. No. 2006/0024298; U.S. Pat. No. 5,457,035); leucine zipper (e.g. from nuclear transforming proteins fos and jun or the proto-oncogene c-myc or from General Control of Nitrogen (GCN4)) (e.g., Busch and Sassone-Corsi (1990) Trends Genetics, 6:36-40; Gentz et al., (1989) Science, 243:1695-1699); a hydrophobic region; a hydrophilic region; or a free thiol which forms an intermolecular disulfide bond between the chimeric molecules of a homo- or heteromultimer. In addition, a multimerization domain can include an amino acid sequence comprising a protuberance complementary to an amino acid sequence comprising a hole, such as is described, for example, in U.S. Pat. No. 5,731,168; International Patent Pub. Nos. WO 98/50431 and WO 2005/063816; Ridgway et al. (1996) Protein Engineering, 9:617-621. Such a multimerization region can be engineered such that steric interactions not only promote stable interaction, but further promote the formation of heterodimers over homodimers from a mixture of chimeric monomers. Generally, protuberances are constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan). Compensatory cavities of identical or similar size to the protuberances are optionally created on the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine). Exemplary multimerization domains are described below.

The variant PD-L2 polypeptide can be joined anywhere, but typically via its N- or C-terminus, to the N- or C-terminus of a multimerization domain to form a chimeric polypeptide. The linkage can be direct or indirect via a linker. The chimeric polypeptide can be a fusion protein or can be formed by chemical linkage, such as through covalent or non-covalent interactions. For example, when preparing a chimeric polypeptide containing a multimerization domain, nucleic acid encoding all or part of a variant PD-L2 polypeptide can be operably linked to nucleic acid encoding the multimerization domain sequence, directly or indirectly or optionally via a linker domain. In some cases, the construct encodes a chimeric protein where the C-terminus of the variant PD-L2 polypeptide is joined to the N-terminus of the multimerization domain. In some instances, a construct can encode a chimeric protein where the N-terminus of the variant PD-L2 polypeptide is joined to the N- or C-terminus of the multimerization domain.

A polypeptide multimer contains multiple, such as two, chimeric proteins created by linking, directly or indirectly, two of the same or different variant PD-L2 polypeptides directly or indirectly to a multimerization domain. In some examples, where the multimerization domain is a polypeptide, a gene fusion encoding the variant PD-L2 polypeptide and multimerization domain is inserted into an appropriate expression vector. The resulting chimeric or fusion protein can be expressed in host cells transformed with the recombinant expression vector, and allowed to assemble into multimers, where the multimerization domains interact to form multivalent polypeptides. Chemical linkage of multimerization domains to variant PD-L2 polypeptides can be effected using heterobifunctional linkers.

The resulting chimeric polypeptides, such as fusion proteins, and multimers formed therefrom, can be purified by any suitable method such as, for example, by affinity chromatography over Protein A or Protein G columns. Where two nucleic acid molecules encoding different polypeptides are transformed into cells, formation of homo- and heterodimers will occur. Conditions for expression can be adjusted so that heterodimer formation is favored over homodimer formation.

In some embodiments, the multimerization domain is an Fc domain or portions thereof from an immunoglobulin. In some embodiments, the immunomodulatory protein comprises a variant PD-L2 polypeptide attached to an immunoglobulin Fc (yielding an “immunomodulatory Fc fusion,” such as a “PD-L2-Fc variant fusion,” also termed a PD-L2 vIgD-Fc fusion). In some embodiments, the attachment of the variant PD-L2 polypeptide is at the N-terminus of the Fc. In some embodiments, the attachment of the variant PD-L2 polypeptide is at the C-terminus of the Fc. In some embodiments, two or more PD-L2 variant polypeptides (the same or different) are independently attached at the N-terminus and at the C-terminus.

In some embodiments, the Fc is murine or human Fc. In some embodiments, the Fc is a mammalian or human IgG1, lgG2, lgG3, or lgG4 Fc regions. In some embodiments, the Fc is derived from IgG1, such as human IgG1. In some embodiments, the Fc comprises the amino acid sequence set forth in SEQ ID NO: 211 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 211.

In some embodiments, the Fc region contains one more modifications to alter (e.g. reduce) one or more of its normal functions. In general, the Fc region is responsible for effector functions, such as complement-dependent cytotoxicity (CDC) and antibody-dependent cell cytotoxicity (ADCC), in addition to the antigen-binding capacity, which is the main function of immunoglobulins. In some cases, effector functions of an Fc region can include programmed cell death and cellular phagocytosis. Additionally, the FcRn sequence present in the Fc region plays the role of regulating the IgG level in serum by increasing the in vivo half-life by conjugation to an in vivo FcRn receptor. In some embodiments, such functions can be reduced or altered in an Fc for use with the provided Fc fusion proteins.

In some embodiments, one or more amino acid modifications may be introduced into the Fc region of a PD-L2-Fc variant fusion provided herein, thereby generating an Fc region variant. In some embodiments, the Fc region variant has decreased effector function. There are many examples of changes or mutations to Fc sequences that can alter effector function. For example, WO 00/42072, WO2006019447, WO2012125850, WO2015/107026, US2016/0017041 and Shields et al. J Biol. Chem. 9(2): 6591-6604 (2001) describe exemplary Fc variants with improved or diminished binding to FcRs. The contents of those publications are specifically incorporated herein by reference.

In some embodiments, the provided variant PD-L2-Fc fusions comprise an Fc region that exhibits reduced effector functions, which makes it a desirable candidate for applications in which the half-life of the PD-L2-Fc variant fusion in vivo is important yet certain effector functions (such as CDC and ADCC) are unnecessary or deleterious. In vitro and/or in vivo cytotoxicity assays can be conducted to confirm the reduction/depletion of CDC and/or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the PD-L2-Fc variant fusion lacks FcγR binding (hence likely lacking ADCC activity), but retains FcRn binding ability. The primary cells for mediating ADCC, NK cells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest is described in U.S. Pat. No. 5,500,362 (see, e.g. Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); U.S. Pat. No. 5,821,337 (see Bruggemann, M. et al., J Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods may be employed (see, for example, ACTI™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, Calif.; and CytoTox 96™ non-radioactive cytotoxicity assay (Promega, Madison, Wis.). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays may also be carried out to confirm that the PD-L2-Fc variant fusion is unable to bind C1q and hence lacks CDC activity. See, e.g., C1q and C3c binding ELISA in WO 2006/029879 and WO 2005/100402. To assess complement activation, a CDC assay may be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, M. S. et al., Blood 101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance/half life determinations can also be performed using methods known in the art (see, e.g., Petkova, S. B. et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

PD-L2-Fc variant fusions with reduced effector function include those with substitution of one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 by EU numbering (U.S. Pat. No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327 by EU numbering, including the so-called “DANA” Fc mutant with substitution of residues 265 and 297 to alanine (U.S. Pat. No. 7,332,581).

In some embodiments, the Fc region of PD-L2-Fc variant fusions has an Fc region in which any one or more of amino acids at positions 234, 235, 236, 237, 238, 239, 270, 297, 298, 325, and 329 (indicated by EU numbering) are substituted with different amino acids compared to the native Fc region. Such alterations of Fc region are not limited to the above-described alterations, and include, for example, alterations such as deglycosylated chains (N297A and N297Q), IgG1-N297G, IgG1-L234A/L235A, IgG1-L234A/L235E/G237A, IgG1-A325A/A330S/P331S, IgG1-C226S/C229S, IgG1-C226S/C229S/E233P/L234V/L235A, IgG1-E233P/L234V/L235A/G236del/S267K, IgG1-L234F/L235E/P331S, IgG1-S267E/L328F, IgG2-V234A/G237A, IgG2-H268Q/V309L/A330S/A331S, IgG4-L235A/G237A/E318A, and IgG4-L236E described in Current Opinion in Biotechnology (2009) 20 (6), 685-691; alterations such as G236R/L328R, L235G/G236R, N325A/L328R, and N325LL328R described in WO 2008/092117; amino acid insertions at positions 233, 234, 235, and 237 (indicated by EU numbering); and alterations at the sites described in WO 2000/042072.

Certain Fc variants with improved or diminished binding to FcRs are described. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312, WO2006019447 and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).)

In some embodiments, there is provided a PD-L2-Fc variant fusion comprising a variant Fc region comprising one or more amino acid substitutions which increase half-life and/or improve binding to the neonatal Fc receptor (FcRn). Antibodies with increased half-lives and improved binding to FcRn are described in US2005/0014934A1 (Hinton et al.) or WO2015107026. Those antibodies comprise an Fc region with one or more substitutions therein which improve binding of the Fc region to FcRn. Such Fc variants include those with substitutions at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434 by EU numbering, e.g., substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826).

In some embodiments, the Fc region of a PD-L2-Fc variant fusion comprises one or more amino acid substitution E356D and M358L by EU numbering. In some embodiments, the Fc region of a PD-L2-Fc variant fusion comprises one or more amino acid substitutions C220S, C226S and/or C229S by EU numbering. In some embodiments, the Fc region of a PD-L2 variant fusion comprises one or more amino acid substitutions R292C and V302C. See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Pat. Nos. 5,648,260; 5,624,821; and WO 94/29351 concerning other examples of Fc region variants.

In some embodiments, alterations are made in the Fc region that result in diminished C1q binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99/51642, and Idusogie et al., J. Immunol. 164: 4178-4184 (2000).

In some embodiments, there is provided a PD-L2-Fc variant fusion comprising a variant Fc region comprising one or more amino acid modifications, wherein the variant Fc region is derived from IgG1, such as human IgG1. In some embodiments, the variant Fc region is derived from the amino acid sequence set forth in SEQ ID NO: 211. In some embodiments, the Fc contains at least one amino acid substitution that is N82G by numbering of SEQ ID NO: 211 (corresponding to N297G by EU numbering). In some embodiments, the Fc further contains at least one amino acid substitution that is R77C or V87C by numbering of SEQ ID NO: 211 (corresponding to R292C or V302C by EU numbering). In some embodiments, the variant Fc region further comprises a C5S amino acid modification by numbering of SEQ ID NO: 211 (corresponding to C220S by EU numbering). For example, in some embodiments, the variant Fc region comprises the following amino acid modifications: V297G and one or more of the following amino acid modifications C220S, R292C or V302C by EU numbering (corresponding to N82G and one or more of the following amino acid modifications C5S, R77C or V87C with reference to SEQ ID NO:211), e.g. the Fc region comprises the sequence set forth in SEQ ID NO:1205. In some embodiments, the variant Fc region comprises one or more of the amino acid modifications C220S, L234A, L235E or G237A, e.g. the Fc region comprises the sequence set forth in SEQ ID NO:1206. In some embodiments, the variant Fc region comprises one or more of the amino acid modifications C220S, L235P, L234V, L235A, G236del or S267K, e.g. the Fc region comprises the sequence set forth in SEQ ID NO:1207. In some embodiments, the variant Fc comprises one or more of the amino acid modifications C220S, L234A, L235E, G237A, E356D or M358L, e.g. the Fc region comprises the sequence set forth in SEQ ID NO:1189.

In some embodiments, the Fc region lacks the C-terminal lysine corresponding to position 232 of the wild-type or unmodified Fc set forth in SEQ ID NO: 211 (corresponding to K447del by EU numbering). In some aspects, such an Fc region can additionally include one or more additional modifications, e.g. amino acid substitutions, such as any as described. Exemplary of such an Fc region is set forth in SEQ ID NO: 1737, 1738, 1739 or 1740.

In some embodiments, there is provided a PD-L2-Fc variant fusion comprising a variant Fc region in which the variant Fc comprises the sequence of amino acids set forth in any of SEQ ID NOS:1189, 1205, 1206, 1207, 1737, 1738, 1739 or 1740 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOS: 1189, 1205, 1206, 1207, 1737, 1738, 1739 or 1740.

In some embodiments, the Fc is derived from IgG2, such as human IgG2. In some embodiments, the Fc comprises the amino acid sequence set forth in SEQ ID NO: 212 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 212.

In some embodiments, the IgG4 Fc is a stabilized Fc in which the CH3 domain of human IgG4 is substituted with the CH3 domain of human IgG1 and which exhibits inhibited aggregate formation, an antibody in which the CH3 and CH2 domains of human IgG4 are substituted with the CH3 and CH2 domains of human IgG1, respectively, or an antibody in which arginine at position 409 indicated in the EU index proposed by Kabat et al. of human IgG4 is substituted with lysine and which exhibits inhibited aggregate formation (see e.g. U.S. Pat. No. 8,911,726. In some embodiments, the Fc is an IgG4 containing the S228P mutation, which has been shown to prevent recombination between a therapeutic antibody and an endogenous IgG4 by Fab-arm exchange (see e.g. Labrijin et al. (2009) Nat. Biotechnol., 27(8)767-71.) In some embodiments, the Fc comprises the amino acid sequence of hIgG4.

In some embodiments, the variant PD-L2 polypeptide is directly linked to the Fc sequence. In some embodiments, the variant PD-L2 polypeptide is indirectly linked to the Fc sequence, such as via a linker. In some embodiments, one or more “peptide linkers” link the variant PD-L2 polypeptide and the Fc domain. In some embodiments, a peptide linker can be a single amino acid residue or greater in length. In some embodiments, the peptide linker has at least one amino acid residue but is no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues in length. In some embodiments, the linker is three alanines (AAA). In some embodiments, the linker is a flexible linker. In some embodiments, the linker is (in one-letter amino acid code): GGGGS (“4GS” or “G₄S”; SEQ ID NO:1742) or multimers of the 4GS linker, such as repeats of 2, 3, 4, or 5 4GS linkers, such as set forth in SEQ ID NO: 264 (2×GGGGS) or SEQ ID NO: 263 (3×GGGGS). In some embodiments, the linker (in one-letter amino acid code) is GSGGGGS (SEQ ID NO:1741). In some embodiments, the linker also can include a series of alanine residues alone or in addition to another peptide linker (such as a 4GS linker or multimer thereof). In some embodiments, the number of alanine residues in each series is: 2, 3, 4, 5, or 6 alanines. In some embodiments, the linker is a rigid linker. For example, the linker is an a-helical linker. In some embodiments, the linker is (in one-letter amino acid code): EAAAK or multimers of the EAAAK linker, such as repeats of 2, 3, 4, or 5 EAAAK linkers, such as set forth in SEQ ID NO: 2030 (1×EAAAK), SEQ ID NO: 2031 (3×EAAAK) or SEQ ID NO: 2032 (5×EAAAK). In some embodiments, the linker can further include amino acids introduced by cloning and/or from a restriction site, for example the linker can include the amino acids GS (in one-letter amino acid code) as introduced by use of the restriction site BAMHI. For example, in some embodiments, the linker (in one-letter amino acid code) is GSGGGGS (SEQ ID NO:1741), GS(G₄S)₃ (SEQ ID NO: 2040), or GS(G₄S)₅ (SEQ ID NO: 2041). In some examples, the linker is a 2×GGGGS followed by three alanines (GGGGSGGGGSAAA; SEQ ID NO: 265). In some cases, the immunomodulatory polypeptide comprising a variant PD-L2 comprises various combinations of peptide linkers.

In some embodiments, the variant PD-L2-Fc fusion protein is a dimer formed by two variant PD-L2 Fc polypeptides linked to an Fc domain. In some embodiments, the dimer is a homodimer in which the two variant PD-L2 Fc polypeptides are the same. In some embodiments, the dimer is a heterodimer in which the two variant PD-L2 Fc polypeptides are different.

Also provided are nucleic acid molecules encoding the variant PD-L2-Fc fusion protein. In some embodiments, for production of an Fc fusion protein, a nucleic acid molecule encoding a variant PD-L2-Fc fusion protein is inserted into an appropriate expression vector. The resulting variant PD-L2-Fc fusion protein can be expressed in host cells transformed with the expression where assembly between Fc domains occurs by interchain disulfide bonds formed between the Fc moieties to yield dimeric, such as divalent, variant PD-L2-Fc fusion proteins.

The resulting Fc fusion proteins can be easily purified by affinity chromatography over Protein A or Protein G columns. For the generation of heterodimers, additional steps for purification can be necessary. For example, where two nucleic acids encoding different variant PD-L2 polypeptides are transformed into cells, the formation of heterodimers must be biochemically achieved since variant PD-L2 molecules carrying the Fc-domain will be expressed as disulfide-linked homodimers as well. Thus, homodimers can be reduced under conditions that favor the disruption of interchain disulfides, but do no effect intra-chain disulfides. In some cases, different variant-PD-L2 Fc monomers are mixed in equimolar amounts and oxidized to form a mixture of homo- and heterodimers. The components of this mixture are separated by chromatographic techniques. Alternatively, the formation of this type of heterodimer can be biased by genetically engineering and expressing Fc fusion molecules that contain a variant PD-L2 polypeptide using knob-into-hole methods described below.

B. Stack Molecules with Additional IgSF Domains

In some embodiments, the immunomodulatory proteins can contain any of the variant PD-L2 polypeptides provided herein linked, directly or indirectly, to one or more other immunoglobulin superfamily (IgSF) domain (“stacked” immunomodulatory protein construct and also called a “Type II” immunomodulatory protein). In some aspects, this can create unique multi-domain immunomodulatory proteins that bind two or more, such as three or more, cognate binding partners, thereby providing a multi-targeting modulation of the immune synapse.

In some embodiments, an immunomodulatory protein comprises a combination (a “non-wild-type combination”) and/or arrangement (a “non-wild type arrangement” or “non-wild-type permutation”) of a variant PD-L2 domain with one or more other affinity modified and/or non-affinity modified IgSF domain sequences of another IgSF family member (e.g. a mammalian IgSF family member) that are not found in wild-type IgSF family members. In some embodiments, the immunomodulatory protein contains 2, 3, 4, 5 or 6 immunoglobulin superfamily (IgSF) domains, where at least one of the IgSF domain is a variant PD-L2 IgSF domain (vIgD of PD-L2) according to the provided description.

In some embodiments, the sequences of the additional IgSF domains can be a modified IgSF domain that contains one or more amino acid modifications, e.g. substitutions, compared to a wildtype or unmodified IgSF domain. In some embodiments, the IgSF domain can be non-affinity modified (e.g., wild-type) or have been affinity modified. In some embodiments, the unmodified or wild-type IgSF domain can be from mouse, rat, cynomolgus monkey, or human origin, or combinations thereof. In some embodiments, the additional IgSF domains can be an IgSF domain of an IgSF family member set forth in Table 2. In some embodiments, the additional IgSF domain can be an affinity-modified IgSF domain containing one or more amino acid modifications, e.g. substitutions, compared to an IgSF domain contained in an IgSF family member set forth in Table 2.

In some embodiments, the additional IgSF domain is an affinity or non-affinity modified IgSF domain contained in an IgSF family member of a family selected from Signal-Regulatory Protein (SIRP) Family, Triggering Receptor Expressed On Myeloid Cells Like (TREML) Family, Carcinoembryonic Antigen-related Cell Adhesion Molecule (CEACAM) Family, Sialic Acid Binding Ig-Like Lectin (SIGLEC) Family, Butyrophilin Family, B7 family, CD28 family, V-set and Immunoglobulin Domain Containing (VSIG) family, V-set transmembrane Domain (VSTM) family, Major Histocompatibility Complex (MHC) family, Signaling lymphocytic activation molecule (SLAM) family, Leukocyte immunoglobulin-like receptor (LIR), Nectin (Nec) family, Nectin-like (NECL) family, Poliovirus receptor related (PVR) family, Natural cytotoxicity triggering receptor (NCR) family, T cell immunoglobulin and mucin (TIM) family or Killer-cell immunoglobulin-like receptors (KIR) family. In some embodiments, the additional IgSF domains are independently derived from an IgSF protein selected from the group consisting of CD80(B7-1), CD86(B7-2), CD274 (PD-L2, B7-H1), PDCD1LG2(PD-L2, CD273), ICOSLG(B7RP1, CD275, ICOSL, B7-H2), CD276(B7-H3), VTCN1(B7-H4), CD28, CTLA4, PDCD1(PD-1), ICOS, BTLA(CD272), CD4, CD8A(CD8-alpha), CD8B(CD8-beta), LAG3, HAVCR2(TIM-3), CEACAM1, TIGIT, PVR(CD155), PVRL2(CD112), CD226, CD2, CD160, CD200, CD200R1(CD200R), and NC R3 (NKp30).

The first column of Table 2 provides the name and, optionally, the name of some possible synonyms for that particular IgSF member. The second column provides the protein identifier of the UniProtKB database, a publicly available database accessible via the internet at uniprot.org or, in some cases, the GenBank Number. The Universal Protein Resource (UniProt) is a comprehensive resource for protein sequence and annotation data. The UniProt databases include the UniProt Knowledgebase (UniProtKB). UniProt is a collaboration between the European Bioinformatics Institute (EMBL-EBI), the SIB Swiss Institute of Bioinformatics and the Protein Information Resource (PIR) and supported mainly by a grant from the U.S. National Institutes of Health (NIH). GenBank is the NIH genetic sequence database, an annotated collection of all publicly available DNA sequences (Nucleic Acids Research, 2013 January; 41(D1):D36-42). The third column provides the region where the indicated IgSF domain is located. The region is specified as a range where the domain is inclusive of the residues defining the range. Column 3 also indicates the IgSF domain class for the specified IgSF region. Column 4 provides the region where the indicated additional domains are located (signal peptide, S; extracellular domain, E; transmembrane domain, T; cytoplasmic domain, C). It is understood that description of domains can vary depending on the methods used to identify or classify the domain, and may be identified differently from different sources. The description of residues corresponding to a domain in Table 2 is for exemplification only and can be several amino acids (such as one, two, three or four) longer or shorter. Column 5 indicates for some of the listed IgSF members, some of its cognate cell surface binding partners.

TABLE 2 IgSF members according to the present disclosure. NCBI Protein Accession IgSF Member Amino Acid Sequence Number/ Cognate Cell (SEQ ID NO) IgSF UniProtKB IgSF Region Surface Precursor Member Protein & Domain Other Binding (mature (Synonyms) Identifier Class Domains Partners residues) Mature ECD CD80 NP_005182.1 35-135, 35- S: 1-34, CD28, CTLA4, SEQ ID NO: 1 SEQ ID SEQ ID (B7-1) P33681 138, 37-138 or E: 35-242, PD-L1 (35-288) NO: 213 NO: 28 35-141 IgV, T: 243-263, 145-230 or C: 264-288 154-232 IgC CD86 P42081.2 33-131 IgV, S: 1-23, CD28, CTLA4 SEQ ID NO: 2 SEQ ID SEQ ID (B7-2) 150-225 IgC2 E: 24-247, (24-329) NO: 214 NO: 29 T: 248-268, C: 269-329 CD274 Q9NZQ7.1 24-130 IgV, S: 1-18, PD-1, B7-1 SEQ ID NO: 3 SEQ ID SEQ ID (PD-L1, 133-225 IgC2 E: 19-238, (19-290) NO: 215 NO: 30 B7-H1) T: 239-259, C: 260-290 PDCD1LG2 Q9BQ51.2 21-118 IgV, S: 1-19, PD-1, RGMb SEQ ID NO: 4 SEQ ID SEQ ID (PD-L2, 122-203 IgC2 E: 20-220, (20-273) NO: 216 NO: 31 CD273) T: 221-241, C: 242-273 ICOSLG O75144.2 19-129 IgV, S: 1-18, ICOS, CD28, SEQ ID NO: 5 SEQ ID SEQ ID (B7RP1, 141-227 IgC2 E: 19-256, CTLA4 (19-302) NO: 217 NO: 32 CD275, T: 257-277, ICOSL, B7- C: 278-302 H2) CD276 Q5ZPR3.1 29-139 IgV, S: 1-28, SEQ ID NO: 6 SEQ ID SEQ ID (B7-H3) 145-238 IgC2, E: 29-466, (29-534) NO: 218 NO: 33 243-357 IgV2, T: 467-487, 363-456, 367- C: 488-534 453 IgC2 VTCN1 Q7Z7D3.1 35-146 IgV, S: 1-24, SEQ ID NO: 7 SEQ ID SEQ ID (B7-H4) 153-241 IgV E: 25-259, (25-282) NO: 219 NO: 34 T: 260-280, C: 281-282 CD28 P10747.1 28-137 IgV S: 1-18, B7-1, B7-2, SEQ ID NO: 8 SEQ ID SEQ ID E: 19-152, B7RP1 (19-220) NO: 220 NO: 35 T: 153-179, C: 180-220 CTLA4 P16410.3 39-140 IgV S: 1-35, B7-1, B7-2, SEQ ID NO: 9 SEQ ID SEQ ID E: 36-161, B7RP1 (36-223) NO: 221 NO: 36 T: 162-182, C: 183-223 PDCD1 Q15116.3 35-145 IgV S: 1-20, PD-L2, PD-L2 SEQ ID NO: 10 SEQ ID SEQ ID (PD-1) E: 21-170, (21-288) NO: 222 NO: 37 T: 171-191, C: 192-288 ICOS Q9Y6W8.1 30-132 IgV S: 1-20, B7RP1 SEQ ID NO: 11 SEQ ID SEQ ID E: 21-140, (21-199) NO: 223 NO: 38 T: 141-161, C: 162-199 BTLA Q7Z6A9.3 31-132 IgV S: 1-30, HVEM SEQ ID NO: 12 SEQ ID SEQ ID (CD272) E: 31-157, (31-289) NO: 224 NO: 39 T: 158-178, C: 179-289 CD4 P01730.1 26-125 IgV, S: 1-25, MHC class II SEQ ID NO: 13 SEQ ID SEQ ID 126-203 IgC2, E: 26-396, (26-458) NO: 225 NO: 40 204-317 IgC2, T: 397-418, 317-389, 318- C: 419-458 374 IgC2 CD8A P01732.1 22-135 IgV S: 1-21, E: MHC class I SEQ ID NO: 14 SEQ ID SEQ ID (CD8- 22-182, T: (22-235) NO: 226 NO: 41 alpha) 183-203, C: 204-235 CD8B P10966.1 22-132 IgV S: 1-21, MHC class I SEQ ID NO: 15 SEQ ID SEQ ID (CD8-beta) E: 22-170, (22-210) NO: 227 NO: 42 T: 171-191, C: 192-210 LAG3 P18627.5 37-167 IgV, S: 1-28, MHC class II SEQ ID NO: 16 SEQ ID SEQ ID 168-252 IgC2, E: 29-450, (29-525) NO: 228 NO: 43 265-343 IgC2, T: 451-471, 349-419 IgC2 C: 472-525 HAVCR2 Q8TDQ0.3 22-124 IgV S: 1-21, CEACAM-1, SEQ ID NO: 17 SEQ ID SEQ ID (TIM-3) E: 22-202, phosphatidylserine, (22-301) NO: 229 NO: 44 T: 203-223, Galectin-9, C: 224-301 HMGB1 CEACAM1 P13688.2 35-142 IgV, S: 1-34, TIM-3 SEQ ID NO: 18 SEQ ID SEQ ID 145-232 IgC2, E: 35-428, (35-526) NO: 230 NO: 45 237-317 IgC2, T: 429-452, 323-413 IgC2 C: 453-526 TIGIT Q495A1.1 22-124 IgV S: 1-21, CD155, CD112 SEQ ID NO: 19 SEQ ID SEQ ID E: 22-141, (22-244) NO: 231 NO: 46 T: 142-162, C: 163-244 PVR P15151.2 24-139 IgV, S: 1-20, TIGIT, CD226, SEQ ID NO: 20 SEQ ID SEQ ID (CD155) 145-237 IgC2, E: 21-343, CD96, (21-417) NO: 232 NO: 47 244-328 IgC2 T: 344-367, poliovirus C: 368-417 PVRL2 Q92692.1 32-156 IgV, S: 1-31, TIGIT, CD226, SEQ ID NO: 21 SEQ ID SEQ ID (CD112) 162-256 IgC2, E: 32-360, CD112R (32-538) NO: 233 NO: 48 261-345 IgC2 T: 361-381, C: 382-538 CD226 Q15762.2 19-126 IgC2, S: 1-18, CD155, CD112 SEQ ID NO: 22 SEQ ID SEQ ID 135-239 IgC2 E: 19-254, (19-336) NO: 234 NO: 49 T: 255-275, C: 276-336 CD2 P06729.2 25-128 IgV, S: 1-24, CD58 SEQ ID NO: 23 SEQ ID SEQ ID 129-209 IgC2 E: 25-209, (25-351) NO: 235 NO: 50 T: 210-235, C: 236-351 CD160 O95971.1 27-122 IgV N/A HVEM, MHC SEQ ID NO: 24 SEQ ID SEQ ID family of (27-159) NO: 236 NO: 51 proteins CD200 P41217.4 31-141 IgV, S: 1-30, CD200R SEQ ID NO: 25 SEQ ID SEQ ID 142-232 IgC2 E: 31-232, (31-278) NO: 237 NO: 52 T: 233-259, C: 260-278 CD200R1 Q8TD46.2 53-139 IgV, S: 1-28, CD200 SEQ ID NO: 26 SEQ ID SEQ ID (CD200R) 140-228 IgC2 E: 29-243, (29-325) NO: 238 NO: 53 T: 244-264, C: 265-325 NCR3 O14931.1 19-126 IgC- S: 1-18, B7-H6 SEQ ID NO: 27 SEQ ID SEQ ID (NKp30) like E: 19-135, (19-201) NO: 239 NO: 54 T: 136-156, C: 157-201 VSIG8 Q5VU13 22-141 IgV,1 S: 1-21 VISTA SEQ ID NO: 240 SEQ ID SEQ ID 146-257 E: 22-263 (22-414) NO: 241 NO: 242 IgV2 T: 264-284 C: 285-414

In some embodiments, the provided immunomodulatory proteins, in addition to containing a variant PD-L2 polypeptide, also contains at least 2, 3, 4, 5 or 6 additional immunoglobulin superfamily (IgSF) domains, such as an IgD domain of an IgSF family member set forth in Table 2. In some embodiments, the provided immunomodulatory protein contains at least one additional IgSF domain (e.g. second IgSF domain). In some embodiments, the provided immunomodulatory protein contains at least two additional IgSF domains (e.g. second and third IgSF domain). In some embodiments, the provided immunomodulatory protein contains at least three additional IgSF domains (e.g. second, third and fourth). In some embodiments, the provided immunomodulatory protein contains at least four additional IgSF domains (e.g. second, third, fourth and fifth). In some embodiments, the provided immunomodulatory protein contains at least five additional IgSF domains (e.g. second, third, fourth, fifth and sixth). In some embodiments, the provided immunomodulatory protein contains at least six additional IgSF domains (e.g. second, third, fourth, fifth, sixth and seventh). In some embodiments, each of the IgSF domains in the immunomodulatory protein are different. In some embodiments, at least one of the additional IgSF domain is the same as at least one other IgSF domain in the immunomodulatory protein. In some embodiments, each of the IgSF domains is from or derived from a different IgSF family member. In some embodiments, at least two of the IgSF domains is from or derived from the same IgSF family member.

In some embodiments, the additional IgSF domain comprises an IgV domain or an IgC (e.g., IgC2) domain or domains, or a specific binding fragment of the IgV domain or a specific binding fragment of the IgC (e.g., IgC2) domain or domains. In some embodiments, the additional IgSF domain is or comprises a full-length IgV domain. In some embodiments, the additional IgSF domain is or comprises a full-length IgC (e.g., IgC2) domain or domains. In some embodiments, the additional IgSF domain is or comprises a specific binding fragment of the IgV domain. In some embodiments, the additional IgSF domain is or comprises a specific binding fragment of the IgC (e.g., IgC2) domain or domains. In some embodiments, the immunomodulatory protein contains at least two additional IgSF domains from a single (same) IgSF member. For example, in some aspects, the immunomodulatory protein contains an ECD or portion thereof of an IgSF member containing a full-length IgV domain and a full-length IgC (e.g., IgC2) domain or domains or specific binding fragments thereof. In some embodiments, the provided immunomodulatory proteins contains at least one additional IgSF domain (e.g. a second or, in some cases, also a third IgSF domain) in which at least one additional or second IgSF domain is an IgSF domain set forth in a wild-type or unmodified IgSF domain or a specific binding fragment thereof contained in the sequence of amino acids set forth in any of SEQ ID NOS: 1-27 and 240. In some embodiments, the wild-type or unmodified IgSF domain is an IgV domain or an IgC domain, such as an IgC1 or IgC2 domain.

In some embodiments, the provided immunomodulatory proteins, in addition to containing a variant PD-L2 polypeptide, also contains at least one additional affinity-modified IgSF domain (e.g. a second or, in some cases, also a third affinity-modified IgSF domain and so on) in which at least one additional IgSF domain is a vIgD that contains one or more amino acid modifications (e.g. substitution, deletion or mutation) compared to an IgSF domain in a wild-type or unmodified IgSF domain, such as an IgSF domain in an IgSF family member set forth in Table 2. In some embodiments, the additional or second affinity-modified IgSF domain comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a wild-type or unmodified IgSF domain or a specific binding fragment thereof contained in the sequence of amino acids set forth in any of SEQ ID NOS: 1-27 and 240. In some embodiments, the wild-type or unmodified IgSF domain is an IgV domain or an IgC domain, such as an IgC1 or IgC2 domain. In some embodiments, the additional, e.g., or second or third IgSF domain is an affinity-modified IgV domain and/or IgC domain. In some embodiments, the one or more additional IgSF domain is an affinity-modified IgSF domain that contains an IgV domain and/or an IgC (e.g., IgC2) domain or domains, or a specific binding fragment of the IgV domain and/or a specific binding fragment of the IgC (e.g., IgC2) domain or domains, in which the IgV and/or IgC domain contains the amino acid modification(s) (e.g., substitution(s)). In some embodiments, the one or more additional affinity-modified IgSF domain contains an IgV domain containing the amino acid modification(s) (e.g. substitution(s)). In some embodiments, the one or more additional affinity-modified IgSF domain include IgSF domains present in the ECD or a portion of the ECD of the corresponding unmodified IgSF family member, such as a full-length IgV domain and a full-length IgC (e.g., IgC2) domain or domains, or specific binding fragments thereof, in which one or both of the IgV and IgC contain the amino acid modification(s) (e.g. substitution(s)).

In some embodiments, the immunomodulatory polypeptide comprising a variant PD-L2 can include one or more vIgD of PD-L2 provided herein. In some embodiments, a variant PD-L2 immunomodulatory protein provided herein will comprise exactly 1, 2, 3, 4, 5 or more variant PD-L2 sequences. In some embodiments, at least two of variant PD-L2 sequences are identical variant IgSF domains.

In some embodiments, the provided immunomodulatory polypeptide comprises two or more vIgD sequences of PD-L2. Multiple variant PD-L2 within the polypeptide chain can be identical (i.e., the same species) to each other or be non-identical (i.e., different species) variant PD-L2 sequences. In addition to single polypeptide chain embodiments, in some embodiments two, three, four, or more of the polypeptides of the invention can be covalently or non-covalently attached to each other. Thus, monomeric, dimeric, and higher order (e.g., 3, 4, 5, or more) multimeric proteins are provided herein. For example, in some embodiments exactly two polypeptides of the invention can be covalently or non-covalently attached to each other to form a dimer. In some embodiments, attachment is made via interchain cysteine disulfide bonds. Compositions comprising two or more polypeptides of the invention can be of an identical species or substantially identical species of polypeptide (e.g, a homodimer) or of non-identical species of polypeptides (e.g., a heterodimer). A composition having a plurality of linked polypeptides of the invention can, as noted above, have one or more identical or non-identical variant PD-L2 of the invention in each polypeptide chain. In some specific embodiments, identical or substantially identical species (allowing for 3 or fewer N-terminus or C-terminus amino acid sequence differences) of PD-L2-Fc variant fusion polypeptides will be dimerized to create a homodimer. Alternatively, different species of PD-L2-Fc variant fusion polypeptides can be dimerized to yield a heterodimer.

In some embodiments, the provided immunomodulatory protein contains at least one additional (e.g., second or, in some cases, also a third IgSF domain and so on) IgSF domain that is a vIgD that contains one or more amino acid substitutions compared to an IgSF domain (e.g. IgV) of a wild-type or unmodified IgSF domain other than PD-L2.

In some embodiments, the one or more additional IgSF domain (e.g. second or third IgSF) domain is an IgSF domain (e.g. IgV) of another IgSF family member that itself also binds to an inhibitory receptor. In some aspects, the one or more additional IgSF domain (e.g. second or third IgSF) domain is an affinity-modified IgSF domain that is a variant IgSF domain (vIgD) of an IgSF family member that bind to an inhibitory receptor and that contains or is a vIgD thereof that contains one or more amino acid substitutions in an IgSF domain (e.g. IgV), in which, in some cases, the one or more amino acid modifications result in increased binding to the inhibitory receptor. In some embodiments, the vIgD contains one or more amino acid modifications (e.g. substitutions, deletions or additions) in a wild-type or unmodified IgSF domain (e.g. IgV) of an IgSF family member that binds to an inhibitory receptor. In addition to PD-1, exemplary of such inhibitory receptors are CTLA-4, LAG3, TIGIT, TIM-3, or BTLA. In some embodiments, the one or more additional IgSF domain is from an IgSF family member selected from CD112, CD155, PD-L1, CD80 or CEACAM1. Thus, in some aspects, provided are multi-target checkpoint antagonists that target or block activity of more than one inhibitory receptor. In some embodiments, the immunomodulatory protein in a multi-target checkpoint antagonist that targets or blocks activity of at least two, three, four or more inhibitory receptors.

In some embodiments, there is provided an immunomodulatory protein containing any one of the variant PD-L2 polypeptides and one or more IgSF domain of an inhibitory receptor, such as a wild-type or unmodified inhibitory receptor. In some embodiments, there is provided an immunomodulatory protein containing any one of the variant PD-L2 polypeptides and one or more IgSF domain of CD80, e.g. wild-type or unmodified CD80, such as an IgV domain set forth in SEQ ID NO:1039, 1113, 2039 or an ECD or a portion thereof (containing the IgV and IgC domain or specific binding fragments thereof) set forth in SEQ ID NO:28 or a portion thereof. In some embodiments, there is provided an immunomodulatory protein containing any one of the variant PD-L2 polypeptides and one or more IgSF domain of PD-L1, e.g. wild-type or unmodified PD-L1, such as an IgV domain set forth in SEQ ID NO: 1258 or 1454 or an ECD or a portion thereof (containing the IgV and IgC domain or specific binding fragments thereof) set forth in SEQ ID NO: 30 or 1812 or a portion thereof. In some embodiments, there is provided an immunomodulatory protein containing any one of the variant PD-L2 polypeptides and one or more IgSF domain of CD112, e.g. wild-type or unmodified CD112, such as an IgV domain set forth in SEQ ID NO: 700 or 795 or an ECD or a portion thereof (containing the IgV and IgC domain or specific binding fragments thereof) set forth in SEQ ID NO:48 or a portion thereof. In some embodiments, there is provided an immunomodulatory protein containing any one of the variant PD-L2 polypeptides and one or more IgSF domain of CD155, e.g. wild-type or unmodified CD155, such as an IgV domain set forth in SEQ ID NO:344 or 387 or an ECD or a portion thereof (containing the IgV and IgC domain or specific binding fragments thereof) set forth in SEQ ID NO:47 or a portion thereof.

In some embodiments, there is provided an immunomodulatory protein containing one or more additional IgSF domain (e.g., second or third IgSF) that is a vIgD of an IgSF family member that binds to an inhibitory receptor in which the one or more amino acid modifications in an IgSF domain (e.g. IgV) results in increased binding affinity of the vIgD, or a fusion or immunomodulatory protein containing the vIgD, for its inhibitory receptor cognate binding partner compared to the unmodified IgSF domain, such as binding affinity that is increased more than 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold 40-fold or 50-fold. In some embodiments, the one or more amino acid modifications in an IgSF domain (e.g. IgV) results in increased selectivity of the vIgD, or a fusion or immunomodulatory protein containing the vIgD for its inhibitory receptor compared to the unmodified IgSF domain. In some embodiments, the increased selectivity is a greater ratio of binding of the vIgD for the inhibitory receptor versus another cognate binding partner, such as a cognate binding partner that is not an inhibitory receptor, compared to the ratio of binding of the unmodified IgSF for the inhibitory receptor versus the another cognate binding partner. In some embodiments, the ratio is greater by at least or at least about 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold 40-fold or 50-fold.

In some embodiments, the at least one additional (e.g. second or third) vIgD is an IgSF domain (e.g. IgV) of a variant CD80 polypeptide that contains one or more amino acid modifications (e.g. substitutions, deletions or additions) in the IgSF domain (e.g. IgV) compared to unmodified or wild-type of CD80, which, in some aspects, result in increased, binding to the inhibitory receptor CTLA-4. Exemplary amino acid modifications, such as substitutions, deletions or additions, in an IgSF domain (e.g. IgV or ECD containing IgV and IgC) of a variant CD80 polypeptide are set forth in Table 3. In some embodiments, there is provided an immunomodulatory protein containing any of the provided variant PD-L2 polypeptides and a variant CD80 polypeptide containing an IgV domain including any of the amino acid modifications set forth in Table 3, such as the IgV domain set forth in any of SEQ ID NOS: 1040-1072, 1074-1112, 1114-1146, 1148-1186 or an IgV domain that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% to any of SEQ ID NOS: 1040-1072, 1074-1112, 1114-1146, 1148-1186 and contains the one or more amino acid modifications. In some embodiments, there is provided an immunomodulatory protein containing any of the provided variant PD-L2 polypeptides and a variant CD80 polypeptide containing an ECD or a portion thereof containing the IgV and/or IgC domains, in which is contained any of the amino acid modifications set forth in Table 3, such as the ECD set forth in any of SEQ ID NOS: 966-998, 1000-1038 or an ECD that contains at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% to any of SEQ ID NOS: 966-998, 1000-1038 and contains the one or more amino acid modifications.

In some embodiments, the at least one additional (e.g., second or third) vIgD is an IgSF domain (e.g. IgV) of a variant CD112 polypeptide that contains one or more amino acid modifications (e.g., substitutions, deletions or additions) in the IgSF domain (e.g., IgV) compared to unmodified or wild-type CD112, which, in some aspects, result in increased binding to the inhibitory receptor TIGIT. Exemplary amino acid modifications, such as substitutions, deletions or additions, in an IgSF domain (e.g. IgV or ECD containing IgV and IgC) of a variant CD112 polypeptide are set forth in Table 4. In some embodiments, there is provided an immunomodulatory protein containing any of the provided variant PD-L2 polypeptides and a variant CD112 polypeptide containing an IgV domain including any of the amino acid modifications set forth in Table 4, such as the IgV domain set forth in any of SEQ ID NOS: 748-794, 796-842, 884-965, 1479-1526 or an IgV domain that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% to any of SEQ ID NOS: 748-794, 796-842, 884-965, 1479-1526, and contains the one more amino acid modifications. In some embodiments, there is provided an immunomodulatory protein containing any of the provided variant PD-L2 polypeptides and a variant CD112 polypeptide containing an ECD or a portion thereof containing the IgV and/or IgC domains, in which is contained any of the amino acid modifications set forth in Table 4, such as the ECD set forth in any of SEQ ID NOS: 701-747, 843-883, 1455-1478 or an ECD that contains at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% to any of SEQ ID NOS: 701-747, 843-883, 1455-1478 and contains the one or more amino acid modifications.

In some embodiments, the at least one additional (e.g., second or third) vIgD is an IgSF domain (e.g. IgV) of a variant CD155 polypeptide that contains one or more amino acid modifications (e.g., substitutions, deletions or additions) in the IgSF domain (e.g., IgV) compared to unmodified or wild-type CD155, which, in some aspects, result in increased binding to the inhibitory receptor TIGIT. Exemplary amino acid modifications, such as substitutions, deletions or additions, in an IgSF domain (e.g. IgV or ECD containing IgV and IgC) of a variant CD155 polypeptide are set forth in Table 5. In some embodiments, there is provided an immunomodulatory protein containing any of the provided variant PD-L2 polypeptides and a variant CD155 polypeptide containing an IgV domain including any of the amino acid modifications set forth in Table 5, such as the IgV domain set forth in any of SEQ ID NOS: 366-386, 388-408, 506-699, 1527-1595, 1597-1598, 1645-1736 or an IgV domain that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% to any of SEQ ID NOS: 366-386, 388-408, 506-699, 1527-1595, 1597-1598, 1645-1736 and contains the one more amino acid modifications. In some embodiments, there is provided an immunomodulatory protein containing any of the provided variant PD-L2 polypeptides and a variant CD155 polypeptide containing an ECD or a portion thereof containing the IgV and/or IgC domains, in which is contained any of the amino acid modifications set forth in Table 5, such as the ECD set forth in any of SEQ ID NOS: 1573-1596, 1599-1644 or an ECD that contains at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% to any of SEQ ID NOS: 1573-1596, 1599-1644 and contains the one or more amino acid modifications.

In some embodiments, the at least one additional (e.g., second or third) vIgD is an IgSF domain (e.g. IgV) of a variant PD-L1 polypeptide that contains one or more amino acid modifications (e.g., substitutions, deletions or additions) in the IgSF domain (e.g., IgV or ECD) compared to unmodified or wild-type PD-L1, which, in some aspects, result in increased binding to the inhibitory receptor PD-1. Exemplary amino acid modifications, such as substitutions, deletions or additions, in an IgSF domain (e.g. IgV or ECD containing IgV and IgC) of a variant PD-L1 polypeptide are set forth in Table 8. In some embodiments, there is provided an immunomodulatory protein containing any of the provided variant PD-L2 polypeptides and a variant PD-L1 polypeptide containing an IgV domain including any of the amino acid modifications set forth in Table 8, such as the IgV domain set forth in any of SEQ ID NOS: 1324-1453, 1810-1811, 1992-2021, or an IgV domain that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% to any of SEQ ID NOS: 1324-1453, 1810-1811, 1992-2021, and contains the one more amino acid modifications. In some embodiments, there is provided an immunomodulatory protein containing any of the provided variant PD-L2 polypeptides and a variant PD-L1 polypeptide containing an ECD or a portion thereof containing the IgV and/or IgC domains, in which is contained any of the amino acid modifications set forth in Table 8, such as the ECD set forth in any of SEQ ID NOS: 1259-1323, 1743-1809, 1813-1991 or an ECD that contains at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% to any of SEQ ID NOS: 1259-1323, 1743-1809, 1813-1991, and contains the one or more amino acid modifications.

In some embodiments, the one or more additional IgSF domain (e.g. second or third IgSF) domain is an IgSF domain (e.g. IgV) of another IgSF family member that binds or recognizes a tumor antigen. In such embodiments, the IgSF family member serves as a tumor-localizing moiety, thereby bringing the vIgD of PD-L2 in close proximity to immune cells in the tumor microenvironment. In some embodiments, the additional IgSF domain (e.g. second IgSF) domain is an IgSF domain of NKp30, which binds or recognizes B7-H6 expressed on a tumor cell. In some embodiments, the at least one additional (e.g. second) IgSF domain, e.g. NKp30, is an affinity-modified IgSF domain or vIgD that contains one or more amino acid modifications (e.g. substitutions, deletions or additions). In some embodiments, the one or more amino acid modifications increase binding affinity and/or selectivity to B7-H6 compared to unmodified IgSF domain, e.g. NKp30, such as by at least or at least about 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold 40-fold or 50-fold. Exemplary amino acid modifications, such as substitutions, deletions or additions, in an IgSF domain (e.g. IgC-like or full ECD) of a variant NKp30 polypeptide are set forth in Table 6. Among the exemplary polypeptides is an NKp30 variant that contains the mutations L30V/A60V/S64P/S86G with reference to positions in the NKp30 extracellular domain corresponding to positions set forth in SEQ ID NO: 54. In some embodiments, there is provided an immunomodulatory protein containing any of the provided variant PD-L2 polypeptides and a variant NKp30 polypeptide containing an IgC-like domain including any of the amino acid modifications set forth in Table 6, such as the IgC-like domain set forth in any of SEQ ID NOS: 1232-1236 or an IgC-like domain that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% to any of SEQ ID NOS: 1232-1236 and contains the one more amino acid modifications. In some embodiments, there is provided an immunomodulatory protein containing any of the provided variant PD-L2 polypeptides and a variant NKp30 polypeptide containing an ECD or a portion thereof containing an IgSF domain or domains, in which is contained any of the amino acid modifications set forth in Table 6, such as the ECD set forth in any of SEQ ID NOS: 1226-1230 or an ECD that contains at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% to any of SEQ ID NOS: 1226-1230 and contains the one or more amino acid modifications.

In some embodiments, the at least one additional (e.g., second or third) vIgD is an IgSF domain (e.g. IgV) of a variant CD86 polypeptide that contains one or more amino acid modifications (e.g., substitutions, deletions or additions) in the IgSF domain (e.g., IgV) compared to unmodified or wild-type CD86, which, in some aspects, result in increased binding to its cognate binding partner. Exemplary amino acid modifications, such as substitutions, deletions or additions, in an IgSF domain (e.g. IgV or ECD containing IgV and IgC) of a variant CD86 polypeptide are set forth in Table 7. Among exemplary polypeptides include CD86 variants that contain the mutations Q35H/H90L/Q102H with reference to positions in the CD86 extracellular domain corresponding to positions set forth in SEQ ID NO: 29. In some embodiments, there is provided an immunomodulatory protein containing any of the provided variant PD-L2 polypeptides and a variant CD86 polypeptide containing an IgV domain including any of the amino acid modifications set forth in Table 7, such as the IgV domain set forth in any of SEQ ID NOS: 1244-1247 or an IgV domain that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% to any of SEQ ID NOS: 1244-1247 and contains the one more amino acid modifications. In some embodiments, there is provided an immunomodulatory protein containing any of the provided variant PD-L2 polypeptides and a variant CD86 polypeptide containing an ECD or a portion thereof containing the IgV and/or IgC domains, in which is contained any of the amino acid modifications set forth in Table 7, such as the ECD set forth in any of SEQ ID NOS: 1239-1242 or an ECD that contains at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% to any of SEQ ID NOS: 1239-1242 and contains the one or more amino acid modifications.

Tables 3-8 provide exemplary polypeptides containing one or more affinity-modified IgSF domains that can be used in stack constructs provided herein.

TABLE 3 Exemplary variant CD80 polypeptides ECD SEQ ID IgV SEQ Mutation(s) NO ID NO Wild-type 28 1039, 2039 L70P 966 1040, 1114 I30F/L70P 967 1041, 1115 Q27H/T41S/A71D 968 1042, 1116 I30T/L70R 969 1043, 1117 T13R/C16R/L70Q/A71D 970 1044, 1118 T57I 971 1045, 1119 M43I/C82R 972 1046, 1120 V22L/M38V/M47T/A71D/L85M 973 1047, 1121 I30V/T57I/L70P/A71D/A91T 974 1048, 1122 V22I/L70M/A71D 975 1049, 1123 N55D/L70P/E77G 976 1050, 1124 T57A/I69T 977 1051, 1125 N55D/K86M 978 1052, 1126 L72P/T79I 979 1053, 1127 L70P/F92S 980 1054, 1128 T79P 981 1055, 1129 E35D/M47I/L65P/D90N 982 1056, 1130 L25S/E35D/M47I/D90N 983 1057, 1131 A71D 985 1059, 1133 E81K/A91S 987 1061, 1135 A12V/M47V/L70M 988 1062, 1136 K34E/T41A/L72V 989 1063, 1137 T41S/A71D/V84A 990 1064, 1138 E35D/A71D 991 1065, 1139 E35D/M47I 992 1066, 1140 K36R/G78A 993 1067, 1141 Q33E/T41A 994 1068, 1142 M47V/N48H 995 1069, 1143 M47L/V68A 996 1070, 1144 S44P/A71D 997 1071, 1145 Q27H/M43I/A71D/R73S 998 1072, 1146 E35D/T57I/L70Q/A71D 1000 1074, 1148 M47I/E88D 1001 1075, 1149 M42I/I61V/A71D 1002 1076, 1150 P51A/A71D 1003 1077, 1151 H18Y/M47I/T57I/A71G 1004 1078, 1152 V20I/M47V/T57I/V84I 1005 1079, 1153 V20I/M47V/A71D 1006 1080, 1154 A71D/L72V/E95K 1007 1081, 1155 V22L/E35G/A71D/L72P 1008 1082, 1156 E35D/A71D 1009 1083, 1157 E35D/I67L/A71D 1010 1084, 1158 Q27H/E35G/A71D/L72P/T79I 1011 1085, 1159 T13R/M42V/M47I/A71D 1012 1086, 1160 E35D 1013 1087, 1161 E35D/M47I/L70M 1014 1088, 1162 E35D/A71D/L72V 1015 1089, 1163 E35D/M43L/L70M 1016 1090, 1164 A26P/E35D/M43I/L85Q/E88D 1017 1091, 1165 E35D/D46V/L85Q 1018 1092, 1166 Q27L/E35D/M47I/T57I/L70Q/E88D 1019 1093, 1167 M47V/I69F/A71D/V83I 1020 1094, 1168 E35D/T57A/A71D/L85Q 1021 1095, 1169 H18Y/A26T/E35D/A71D/L85Q 1022 1096, 1170 E35D/M47L 1023 1097, 1171 E23D/M42V/M43I/I58V/L70R 1024 1098, 1172 V68M/L70M/A71D/E95K 1025 1099, 1173 N55I/T57I/I69F 1026 1100, 1174 E35D/M43I/A71D 1027 1101, 1175 T41S/T57I/L70R 1028 1102, 1176 H18Y/A71D/L72P/E88V 1029 1103, 1177 V20I/A71D 1030 1104, 1178 E23G/A26S/E35D/T62N/A71D/L72V/L85M 1031 1105, 1179 A12T/E24D/E35D/D46V/I61V/L72P/E95V 1032 1106, 1180 V22L/E35D/M43L/A71G/D76H 1033 1107, 1181 E35G/K54E/A71D/L72P 1034 1108, 1182 L70Q/A71D 1035 1109, 1183 A26E/E35D/M47L/L85Q 1036 1110, 1184 D46E/A71D 1037 1111, 1185 Y31H/E35D/T41S/V68L/K93R/R94W 1038 1112, 1186

TABLE 4 Exemplary variant CD112 polypeptides ECD SEQ ID IgV SEQ Mutation(s) NO ID NO Wild-type 48 700, 795 Y33H, A112V, G117D 701 748, 796 V19A, Y33H, S64G, S80G, G98S, N106Y, A112V 702 749, 797 L32P, A112V 703 750, 798 A95V, A112I 704 751, 799 P28S, A112V 705 752, 800 P27A, T38N, V101A, A112V 706 753, 801 S118F 707 754, 802 R12W, H48Y, F54S, S118F 708 755, 803 R12W, Q79R, S118F 709 756, 804 T113S, S118Y 710 757, 805 S118Y 711 758, 806 N106I, S118Y 712 759, 807 N106I, S118F 713 760, 808 A95T, L96P, S118Y 714 761, 809 Y33H, P67S, N106Y, A112V 715 762, 810 N106Y, A112V 716 763, 811 T18S, Y33H, A112V 717 764, 812 P9S, Y33H, N47S, A112V 718 765, 813 P42S, P67H, A112V 719 766, 814 P27L, L32P, P42S, A112V 720 767, 815 G98D, A112V 721 768, 816 Y33H, S35P, N106Y, A112V 722 769, 817 L32P, P42S, T100A, A112V 723 770, 818 P27S, P45S, N106I, A112V 724 771, 819 Y33H, N47K, A112V 725 772, 820 Y33H, N106Y, A112V 726 773, 821 K78R, D84G, A112V, F114S 727 774, 822 Y33H, N47K, F54L, A112V 728 775, 823 Y33H, A112V 729 776, 824 A95V, A112V 730 777, 825 R12W, A112V 731 778, 826 R12W, P27S, A112V 732 779, 827 Y33H, V51M, A112V 733 780, 828 Y33H, A112V, S118T 734 781, 829 Y33H, V101A, A112V, P115S 735 782, 830 H24R, T38N, D43G, A112V 736 783, 831 A112V 737 784, 832 P27A, A112V 738 785, 833 A112V, S118T 739 786, 834 R12W, A112V, M122I 740 787, 835 Q83K, N106Y, A112V 741 788, 836 R12W, P27S, A112V, S118T 742 789, 837 P28S, Y33H, A112V 743 790, 838 P27S, Q90R, A112V 744 791, 839 L15V, P27A, A112V, S118T 745 792, 840 Y33H, N106Y, T108I, A112V 746 793, 841 Y33H, P56L, V75M, V101M, A112V 747 794, 842 N47K, Q79R, S118F 843 884, 925 Q40R, P60T, A112V, S118T 844 885, 926 F114Y, S118F 845 886, 927 Y33H, K78R, S118Y 846 887, 928 R12W, A46T, K66M, Q79R, N106I, T113A, S118F 847 888, 929 Y33H, A112V, S118F 848 889, 930 R12W, Y33H, N106I, S118F 849 890, 931 L15V, Q90R, S118F 850 891, 932 N47K, D84G, N106I, S118Y 851 892, 933 L32P, S118F 852 893, 934 Y33H, Q79R, A112V, S118Y 853 894, 935 T18A, N106I, S118T 854 895, 936 L15V, Y33H, N106Y, A112V, S118F 855 896, 937 V37M, S118F 856 897, 938 N47K, A112V, S118Y 857 898, 939 A46T, A112V 858 899, 940 P28S, Y33H, N106I, S118Y 859 900, 941 P30S, Y33H, N47K, V75M, Q79R, N106I, S118Y 860 901, 942 V19A, N47K, N106Y, K116E, S118Y 861 902, 943 Q79R, T85A, A112V, S118Y 862 903, 944 V101M, N106I, S118Y 863 904, 945 Y33H, Q79R, N106I, A112V, S118T 864 905, 946 Q79R, A112V 865 906, 947 Y33H, A46T, Q79R, N106I, S118F 866 907, 948 A112V, G121S 867 908, 949 Y33H, Q79R, N106I, S118Y 868 909, 950 Y33H, N106I, A112V 869 910, 951 Y33H, A46T, V101M, A112V, S118T 870 911, 952 L32P, L99M, N106I, S118F 871 912, 953 L32P, T108A, S118F 872 913, 954 R12W, Q79R, A112V 873 914, 955 Y33H, N106Y, E110G, A112V 874 915, 956 Y33H, N106I, S118Y 875 916, 957 Q79R, S118F 876 917, 958 Y33H, Q79R, G98D, V101M, A112V 877 918, 959 N47K, T81S, V101M, A112V, S118F 878 919, 960 G82S, S118Y 879 920, 961 Y33H, A112V, S118Y 880 921, 962 Y33H, N47K, Q79R, N106Y, A112V 881 922, 963 Y33H, S118T 882 923, 964 R12W, Y33H, Q79R, V101M, A112V 883 924, 965 Y33H, Q83K, A112V, S118T 1455 1479, 1503 V29M, Y33H, N106I, S118F 1456 1480, 1504 Y33H, A46T, A112V 1457 1481, 1505 Y33H, Q79R, S118F 1458 1482, 1506 Y33H, N47K, F74L, S118F 1459 1483, 1507 R12W, V101M, N106I, S118Y 1460 1484, 1508 A46T, V101A, N106I, S118Y 1461 1485, 1509 N106Y, A112V, S118T 1462 1486, 1510 S76P, T81I, V101M, N106Y, A112V, S118F 1463 1487, 1511 P9R, L21V, P22L, I34M, S69F, F74L, A87V, 1464 1488, 1512 A112V, L125A Y33H, V101M, A112V 1465 1489, 1513 V29A, L32P, S118F 1466 1490, 1514 Y33H, V101M, N106I, A112V 1467 1491, 1515 R12W, Y33H, N47K, Q79R, S118Y 1468 1492, 1516 Y33H, A46T, A112V, S118T 1469 1493, 1517 Y33H, A112V, F114L, S118T 1470 1494, 1518 Y33H, T38A, A46T, V101M, A112V 1471 1495, 1519 P28S, Y33H, S69P, N106I, A112V, S118Y 1472 1496, 1520 Y33H, P42L, N47K, V101M, A112V 1473 1497, 1521 Y33H, N47K, F74S, Q83K, N106I, F111L, A112V, 1474 1498, 1522 S118T Y33H, A112V, S118T, V119A 1475 1499, 1523 Y33H, N106I, A112V, S118F 1476 1500, 1524 Y33H, K66M, S118F, W124L 1477 1501, 1525 N106I, A112V 1478 1502, 1526

TABLE 5 Exemplary variant CD155 polypeptides ECD SEQ IgV SEQ Mutation(s) ID NO ID NO Wild-type 47 344, 387 P18S, P64S, F91S 345 366, 388 P18S, F91S, L104P 346 367, 389 L44P 347 368, 390 A56V 348 369, 391 P18L, L79V, F91S 349 370, 392 P18S, F91S 350 371, 393 P18T, F91S 351 372, 394 P18T, S42P, F91S 352 373, 395 G7E, P18T, Y30C, F91S 353 374, 396 P18T, F91S, G111D 354 375, 397 P18S, F91P 355 376, 398 P18T, F91S, F108L 356 377, 399 P18T, T45A, F91S 357 378, 400 P18T, F91S, R94H 358 379, 401 P18S, Y30C, F91S 359 380, 402 A81V, L83P 360 381, 403 L88P 361 382, 404 R94H 362 383, 405 A13E, P18S, A56V, F91S 363 384, 406 P18T, F91S, V115A 364 385, 407 P18T, Q60K 365 386, 408 S52M 409 506, 603 T45Q, S52L, L104E, G111R 410 507, 604 S42G 411 508, 605 Q62F 412 509, 606 S52Q 413 510, 607 S42A, L104Q, G111R 414 511, 608 S42A, S52Q, L104Q, G111R 415 512, 609 S52W, L104E 416 513, 610 S42C 417 514, 611 S52W 418 515, 612 S52M, L104Q 419 516, 613 S42L, S52L, Q62F, L104Q 420 517, 614 S42W 421 518, 615 S42Q 422 519, 616 S52L 423 520, 617 S52R 424 521, 618 L104E 425 522, 619 G111R 426 523, 620 S52E 427 524, 621 Q62Y 428 525, 622 T45Q, S52M, L104E 429 526, 623 S42N, L104Q, G111R 430 527, 624 S52M, V57L 431 528, 625 S42N, S52Q, Q62F 432 529, 626 S42A, S52L, L104E, G111R 433 530, 627 S42W, S52Q, V57L, Q62Y 434 531, 628 L104Q 435 532, 629 S42L, S52Q, L104E 436 533, 630 S42C, S52L 437 534, 631 S42W, S52R, Q62Y, L104Q 438 535, 632 T45Q, S52R, L104E 439 536, 633 S52R, Q62F, L104Q, G111R 440 537, 634 T45Q, S52L, V57L, L104E 441 538, 635 S52M, Q62Y 442 539, 636 Q62F, L104E, G111R 443 540, 637 T45Q, S52Q 444 541, 638 S52L, L104E 445 542, 639 S42V, S52E 446 543, 640 T45Q, S52R, G111R 447 544, 641 S42G, S52Q, L104E, G111R 448 545, 642 S42N, S52E, V57L, L104E 449 546, 643 S42C, S52M, Q62F 450 547, 644 S42L 451 548, 645 S42A 452 549, 646 S42G, S52L, Q62F, L104Q 453 550, 647 S42N 454 551, 648 P18T, S65A, S67V, F91S 455 552, 649 P18F, T39A, T45Q, T61R, S65N, S67L, E73G, R78G 456 553, 650 P18T, T45Q, T61R, S65N, S67L 457 554, 651 P18F, S65A, S67V, F91S 458 555, 652 P18F, T45Q, T61R, S65N, S67L, F91S, L104P 459 556, 653 P18S, L79P, L104M 460 557, 654 P18S, L104M 461 558, 655 L79P, L104M 462 559, 656 P18T, T45Q, L79P 463 560, 657 P18T, T45Q, T61R, S65H, S67H 464 561, 658 P18T, A81E 465 562, 659 P18S, D23Y, E37P, S52G, Q62M, G80S, A81P, G99Y, S112N 466 563, 660 A13R, D23Y, E37P, S42P, Q62Y, A81E 467 564, 661 A13R, D23Y, E37P, G99Y, S112N 468 565, 662 A13R, D23Y, E37P, Q62M, A77V, G80S, A81P, G99Y 469 566, 663 P18L, E37S, Q62M, G80S, A81P, G99Y, S112N 470 567, 664 P18S, L104T 471 568, 665 P18S, Q62H, L79Q, F91S 472 569, 666 T45Q, S52K, Q62F, L104Q, G111R 473 570, 667 T45Q, S52Q, Q62Y, L104Q, G111R 474 571, 668 T45Q, S52Q, Q62Y, L104E, G111R 475 572, 669 V57A, T61M, S65W, S67A, E96D, L104T 476 573, 670 P18L, V57T, T61S, S65Y, S67A, L104T 477 574, 671 P18T, T45Q 478 575, 672 P18L, V57A, T61M, S65W, S67A, L104T 479 576, 673 T61M, S65W, S67A, L104T 480 577, 674 P18S, V41A, S42G, T45G, L104N 481 578, 675 P18H, S42G, T451, S52T, G53R, S54H, V57L, H59E, T61S, S65D, 482 579, 676 E68G, L104N P18S, S42G, T45V, F58L, S67W, L104N 483 580, 677 P18S, T45I, L104N 484 581, 678 P18S, S42G, T45G, L104N, V106A 485 582, 679 P18H, H40R, S42G, T45I, S52T, G53R, S54H, V57L, H59E, T61S, 486 583, 680 S65D, E68G, L104Y, V106L, F108H E37V, S42G, T45G, L104N 487 584, 681 P18S, T45Q, L79P, L104T 488 585, 682 P18L, Q62R 489 586, 683 A13R, D23Y, E37P, S42L, S52G, Q62Y, A81E 490 587, 684 P18L, H49R, L104T, D116N 491 588, 685 A13R, D23Y, E37P, Q62M, G80S, A81P, L104T 492 589, 686 S65T, L104T 493 590, 687 A13R, D23Y, E37P, S52G, V57A, Q62M, K70E, L104T 494 591, 688 P18L, A47V, Q62Y, E73D, L104T 495 592, 689 H40T, V41M, A47V, S52Q, Q62L, S65T, E73R, D97G, E98S, L104T, 496 593, 690 D116N P18L, S42P, T45Q, T61G, S65H, S67E, L104T, D116N 497 594, 691 P18S, H40T, V41M, A47V, S52Q, Q62L, S65T, E73R, L104M, 498 595, 692 V106A H40T, V41M, A47V, S52Q, Q62L, S65T, E68G, E73R, D97G, 499 596, 693 E98S, L104T T45Q, S52E, L104E 500 597, 694 T45Q, S52E, Q62F, L104E 501 598, 695 P18F, T26M, L44V, Q62K, L79P, F91S, L104M, G111D 502 599, 696 P18S, T45S, T61K, S65W, S67A, F91S, G111R 503 600, 697 P18S, L79P, L104M, T107M 504 601, 698 P18S, S65W, S67A, M90V, V95A, L104Q, G111R 505 602, 699 P18S, A47G, L79P, F91S, L104M, T107A, R113W 1573 1527, 1550 P18T, D23G, S24A, N35D, H49L, L79P, F91S, L104M, G111R 1574 1528, 1551 V9L, P18S, Q60R, V75L, L79P, R89K, F91S, L104E, G111R 1575 1529, 1552 P18S, H49R, E73D, L79P, N85D, F91S, V95A, L104M, G111R 1576 1530, 1553 V11A, P18S, L79P, F91S, L104M, G111R 1577 1531, 1554 V11A, P18S, S54R, Q60P, Q62K, L79P, N85D, F91S, T107M 1578 1532, 1555 P18T, S52P, S65A, S67V, L79P, F91S, L104M, G111R 1579 1533, 1556 P18T, M36T, L79P, F91S, G111R 1580 1534, 1557 D8G, P18S, M36I, V38A, H49Q, A76E, F91S, L104M, T107A, 1581 1535, 1558 R113W P18S, S52P, S65A, S67V, L79P, F91S, L104M, T107S, R113W 1582 1536, 1559 T15I, P18T, L79P, F91S, L104M, G111R 1583 1537, 1560 P18F, T26M, L44V, Q62K, L79P, E82D, F91S, L104M, G111D 1584 1538, 1561 P18T, E37G, G53R, Q62K, L79P, F91S, E98D, L104M, T107M 1585 1539, 1562 P18L, K70E, L79P, F91S, V95A, G111R 1586 1540, 1563 V9I, Q12K, P18F, S65A, S67V, L79P, L104T, G111R, S112I 1587 1541, 1564 P18F, S65A, S67V, F91S, L104M, G111R 1588 1542, 1565 V9I, V10I, P18S, F20S, T45A, L79P, F91S, L104M, F108Y, G111R, 1589 1543, 1566 S112V V9L, P18L, L79P, M90I, F91S, T102S, L104M, G111R 1590 1544, 1567 P18C, T26M, L44V, M55I, Q62K, L79P, F91S, L104M, T107M 1591 1545, 1568 V9I, P18T, D23G, L79P, F91S, G111R 1592 1546, 1569 P18F, L79P, M90L, F91S, V95A, L104M, G111R 1593 1547, 1570 P18T, M36T, S65A, S67E, L79Q, A81T, F91S, G111R 1594 1548, 1571 V9L, P18T, Q62R, L79P, F91S, L104M, G111R 1595 1549, 1572 P18S, S65W, S67A, L104Q, G111R 1596 1597, 1598 P18T, G19D, M36T, S54N, L79P, L83Q, F91S, T107M, F108Y 1599 1645, 1691 V9L, P18L, M55V, S69L, L79P, A81E, F91S, T107M 1600 1646, 1692 P18F, H40Q, T61K, Q62K, L79P, F91S, L104M, T107V 1601 1647, 1693 P18S, Q32R, Q62K, R78G, L79P, F91S, T107A, R113W 1602 1648, 1694 Q12H, P18T, L21S, G22S, V57A, Q62R, L79P, F91S, T107M 1603 1649, 1695 V9I, P18S, S24P, H49Q, F58Y, Q60R, Q62K, L79P, F91S, T107M 1604 1650, 1696 P18T, W46C, H49R, S65A, S67V, A76T, L79P, S87T, L104M 1605 1651, 1697 P18S, S42T, E51G, L79P, F91S, G92W, T107M 1606 1652, 1698 V10F, T15S, P18L, R48Q, L79P, F91S, T107M, V115M 1607 1653, 1699 P18S, L21M, Y30F, N35D, R84W, F91S, T107M, D116G 1608 1654, 1700 P18F, E51V, S54G, Q60R, L79Q, E82G, S87T, M90I, F91S, G92R, 1609 1655, 1701 T107M Q16H, P18F, F91S, T107M 1610 1656, 1702 P18T, D23G, Q60R, S67L, L79P, F91S, T107M, V115A 1611 1657, 1703 D8G, V9I, V11A, P18T, T26M, S52P, L79P, F91S, G92A, T107L, 1612 1658, 1704 V115A V9I, P18F, A47E, G50S, E68G, L79P, F91S, T107M 1613 1659, 1705 P18S, M55I, Q62K, S69P, L79P, F91S, T107M 1614 1660, 1706 P18T, T39S, S52P, S54R, L79P, F91S, T107M 1615 1661, 1707 P18S, D23N, L79P, F91S, T107M, S114N 1616 1662, 1708 P18S, P34S, E51V, L79P, F91S, G111R 1617 1663, 1709 P18S, H59N, V75A, L79P, A81T, F91S, L104M, T107M 1618 1664, 1710 P18S, W46R, E68D, L79P, F91S, T107M, R113G 1619 1665, 1711 V9L, P18F, T45A, S65A, S67V, R78K, L79V, F91S, T107M, S114T 1620 1666, 1712 P18T, M55L, T61R, L79P, F91S, V106I, T107M 1621 1667, 1713 T15I, P18S, V33M, N35F, T39S, M55L, R78S, L79P, F91S, T107M 1622 1668, 1714 P18S, Q62K, K70E, L79P, F91S, G92E, R113W 1623 1669, 1715 P18F, F20I, T26M, A47V, E51K, L79P, F91S 1624 1670, 1716 P18T, D23A, Q60H, L79P, M90V, F91S, T107M 1625 1671, 1717 P18S, D23G, C29R, N35D, E37G, M55I, Q62K, S65A, S67G, R78G, 1626 1672, 1718 L79P, F91S, L104M, T107M, Q110R A13E, P18S, M36R, Q62K, S67T, L79P, N85D, F91S, T107M 1627 1673, 1719 V9I, P18T, H49R, L79P, N85D, F91S, L104T, T107M 1628 1674, 1720 V9A, P18F, T61S, Q62L, L79P, F91S, G111R 1629 1675, 1721 D8E, P18T, T61A, L79P, F91S, T107M 1630 1676, 1722 P18S, V41A, H49R, S54C, L79S, N85Y, L88P, F91S, L104M, 1631 1677, 1723 T107M V11E, P18H, F20Y, V25E, N35S, H49R, L79P, F91S, T107M, 1632 1678, 1724 G111R V11A, P18F, D23A, L79P, G80D, V95A, T107M 1633 1679, 1725 P18S, K70R, L79P, F91S, G111R 1634 1680, 1726 V9L, V11M, P18S, N35S, S54G, Q62K, L79P, L104M, T107M, 1635 1681, 1727 V9L, P18Y, V25A, V38G, M55V, A77T, L79P, M90I, F91S, L104M 1636 1682, 1728 V10G, P18T, L72Q, L79P, F91S, T107M 1637 1683, 1729 P18S, H59R, A76G, R78S, L79P 1638 1684, 1730 V9A, P18S, M36T, S65G, L79P, F91S, L104T, G111R, S112I 1639 1685, 1731 P18T, S52A, V57A, Q60R, Q62K, S65C, L79P, F91T, N100Y, 1640 1686, 1732 T107M V11A, P18F, N35D, A47E, Q62K, L79P, F91S, G99D, T107M, 1641 1687, 1733 S114N V11A, P18T, N35S, L79P, S87T, F91S 1642 1688, 1734 V9D, V11M, Q12L, P18S, E37V, M55I, Q60R, K70Q, L79P, F91S, 1643 1689, 1735 L104M, T107M T15S, P18S, Y30H, Q32L, Q62R, L79P, F91S, T107M 1644 1690, 1736

TABLE 6 Exemplary variant NKp30 polypeptides IgC-like ECD domain SEQ SEQ ID ID Mutation(s) NO NO Wild-type  54  1231, 1238 L30V/A60V/S64P/S86G 1226 1232 L30V 1227 1233 A60V 1228 1234 S64P 1229 1235 S86G 1230 1236

TABLE 7 Exemplary variant CD86 polypeptides ECD IgV SEQ ID SEQ ID Mutation(s) NO NO Wild-type 29 1243 Q35H/H9OL/Q102H 1239 1244 Q35H 1240 1245 H90L 1241 1246 Q102H 1242 1247

TABLE 8 Exemplary variant PD-Ll polypeptides ECD IgV SEQ SEQ ID ID Mutation(s) NO NO Wild-type  30, 1812 1258, 1454 K28N/M41V/N45T/H51N/K57E 1259, 1743 1324, 1389 I20L/I36T/N45D/I47T 1260, 1744 1325, 1390 I20L/M41K/K44E 1261, 1745 1326, 1391 P6S/N45T/N78I/I83T 1262, 1746 1327, 1392 N78I 1263, 1747 1328, 1393 M41K/N78I 1264, 1748 1329, 1394 N45T/N78I 1265, 1749 1330, 1395 I20L/N45T 1266, 1750 1331, 1396 N45T 1267, 1751 1332, 1397 M41K 1268, 1752 1333, 1398 I20L/I36T/N45D 1269, 1753 1334, 1399 N17D/N45T/V50A/D72G 1270, 1754 1335, 1400 I20L/F49S 1271, 1755 1336, 1401 N45T/V50A 1272, 1756 1337, 1402 I20L/N45T/N78I 1273, 1757 1338, 1403 I20L/N45T/V50A 1274, 1758 1339, 1404 M41V/N45T 1275, 1759 1340, 1405 M41K/N45T 1276, 1760 1341, 1406 A33D/S75P/D85E 1277, 1761 1342, 1407 M181/M41K/D43G/H51R/N781 1278, 1762 1343, 1408 V11E/I20L/I36T/N45D/H60R/S75P 1279, 1763 1344, 1409 A33D/V50A 1280, 1764 1345, 1410 S16G/A33D/K71E/S75P 1281, 1765 1346, 1411 E27G/N45T/M97I 1282, 1766 1347, 1412 E27G/N45T/K57R 1283, 1767 1348, 1413 A33D/E53V 1284, 1768 1349, 1414 D43G/N45D/V58A 1285, 1769 1350, 1415 E40G/D43V/N45T/V50A 1286, 1770 1351, 1416 Y14S/K28E/N45T 1287, 1771 1352, 1417 A33D/N78S 1288, 1772 1353, 1418 A33D/N78I 1289, 1773 1354, 1419 A33D/N45T 1290, 1774 1355, 1420 A33D/N45T/N78I 1291, 1775 1356, 1421 E27G/N45T/V50A 1292, 1776 1357, 1422 N45T/V50A/N78S 1293, 1777 1358, 1423 I20L/N45T/V110M 1294, 1778 1359, 1424 I20L/I36T/N45T/V50A 1295, 1779 1360, 1425 N45T/L74P/S75P 1296, 1780 1361, 1426 N45T/S75P 1297, 1781 1362, 1427 S75P/K106R 1298, 1782 1363, 1428 S75P 1299, 1783 1364, 1429 A33D/S75P 1300, 1784 1365, 1430 A33D/S75P/D104G 1301, 1785 1366, 1431 A33D/S75P 1302, 1786 1367, 1432 I20L/E27G/N45T/V50A 1303, 1787 1368, 1433 I20L/E27G/D43G/N45D/V58A/N78I 1304, 1788 1369, 1434 I20L/D43G/N45D/V58A/N78I 1305, 1789 1370, 1435 I20L/A33D/D43G/N45D/V58A/N78I 1306, 1790 1371, 1436 I20L/D43G/N45D/N78I 1307, 1791 1372, 1437 E27G/N45T/V50A/N78I 1308, 1792 1373, 1438 N45T/V50A/N78I 1309, 1793 1374, 1439 V11A/I20L/E27G/D43G/N45D/ 1310, 1794 1375, 1440 H51Y/S99G I20L/E27G/D43G/N45T/V50A 1311, 1795 1376, 1441 I20L/K28E/D43G/N45D/V58A/Q89R 1312, 1796 1377, 1442 I20L/I36T/N45D 1313, 1797 1378, 1443 I20L/K28E/D43G/N45D/E53G/ 1314, 1798 1379, 1444 V58A/N78I A33D/D43G/N45D/V58A/S75P 1315, 1799 1380, 1445 K23R/D43G/N45D 1316, 1800 1381, 1446 I20L/D43G/N45D/V58A/N78I/ 1317, 1801 1382, 1447 D90G/G101D D43G/N45D/L56Q/V58A/ 1318, 1802 1383, 1448 G101G-ins(G101GG) I20L/K23E/D43G/N45D/V58A/N78I 1319, 1803 1384, 1449 I20L/K23E/D43G/N45D/V50A/N78I 1320, 1804 1385, 1450 T19I/E27G/N45I/V50A/N78I/M97K 1321, 1805 1386, 1451 I20L/M41K/D43G/N45D 1322, 1806 1387, 1452 K23R/N45T/N78I 1323, 1807 1388, 1453 I20L/K28E/D43G/N45D/V58A/ 1808, 1809 1810, 1811 Q89R/G101G-ins (G101GG) K57R/S99G 1813, 1903 1992, 2007 K57R/S99G/F189L 1814, 1904 M18V/M97L/F193S/R195G/ 1815, 1905 E200K/H202Q I36S/M41K/M97L/K144Q/R195G/ 1816, 1906 E200K/H202Q/L206F C22R/Q65L/L124S/K144Q/R195G/ 1817 E200N/H202Q/T221L M18V/I98L/L124S/P198T/L206F 1818, 1907 S99G/N117S/I148V/K171R/R180S 1819, 1908 I36T/M97L/A103V/Q155H 1820, 1909 K28I/S99G 1821, 1910 1993, 2008 R195S 1822, 1911 A79T/S99G/T185A/R195G/E200K/ 1823, 1912 H202Q/L206F K57R/S99G/L124S/K144Q 1824, 1913 K57R/S99G/R195G 1825, 1914 D55V/M97L/S99G 1826, 1915 1994, 2009 E27G/I36T/D55N/M97L/K111E 1827, 1916 1995, 2010 E54G/M97L/S99G 1828, 1917 1996, 2011 G15A/I36T/M97L/K111E/H202Q 1829, 1918 G15A/I36T/V129D 1830, 1919 G15A/I36T/V129D/R195G 1831, 1920 G15A/V129D 1832, 1921 I36S/M97L 1833, 1922 1997, 2012 136T/D55N/M97L/K111E/A204T 1834, 1923 136T/D55N/M97L/K111E/V129A/F173L 1835, 1924 136T/D55S/M97L/K111E/I148V/R180S 1836, 1925 136T/G52R/M97L/V112A/ 1837, 1926 K144E/V175A/P198T 136T/I46V/D55G/M97L/K106E/ 1838, 1927 K144E/T185A/R195G I36T/I83T/M97L/K144E/P198T 1839, 1928 I36T/M97L/K111E 1840, 1929 1998, 2013 I36T/M97L/K144E/P198T 1841, 1930 I36T/M97L/Q155H/F193S/N201Y 1842, 1931 I36T/M97L/V129D 1843, 1932 L35P/I36S/M97L/K111E 1844, 1933 1999, 2014 M18I/I36T/E53G/M97L/K144E/ 1845, 1934 E199G/V207A M18T/I36T/D55N/M97L/K111E 1846, 1935 2000, 2015 M18V/M97L/T176N/R195G 1847, 1936 M97L/S99G 1848, 1937 2001, 2016 N17D/M97L/S99G 1849, 1938 2002, 2017 S99G/T185A/R195G/P198T 1850, 1939 V129D/H202Q 1851, 1940 V129D/P198T 1852, 1941 V129D/T150A 1853, 1942 V93E/V129D 1854, 1943 Y10F/M18V/S99G/Q138R/T203A 1855, 1944 N45D 1856, 1945 2003, 2018 K160M/R195G 1857, 1946 N45D/K144E 1858, 1947 N45D/P198S 1859, 1948 N45D/P198T 1860, 1949 N45D/R195G 1861, 1950 N45D/R195S 1862, 1951 N45D/S131F 1863, 1952 N45D/V58D 1864, 1953 2004, 2019 V129D/R195S 1865, 1954 I98T/F173Y/L196S 1866, 1955 N45D/E134G/L213P 1867, 1956 N45D/F173I/S177C 1868, 1957 N45D/I148V/R195G 1869, 1958 N45D/K111T/R195G 1870, 1959 N45D/N113Y/R195S 1871, 1960 N45D/N165Y/E170G 1872, 1961 N45D/Q89R/I98V 1873, 1962 2005, 2020 N45D/S131F/P198S 1874, 1963 N45D/S75P/P198S 1875, 1964 N45D/V50A/R195T 1876, 1965 E27D/N45D/T183A/I188V 1877, 1966 F173Y/T183I/L196S/T203A 1878, 1967 K23N/N45D/S75P/N120S 1879, 1968 N45D/G102D/R194W/R195G 1880, 1969 N45D/G52V/Q121L/P198S 1881, 1970 N45D/I148V/R195G/N201D 1882, 1971 N45D/K111T/T183A/I188V 1883, 1972 N45D/Q89R/F189S/P198S 1884, 1973 N45D/S99G/C137R/V207A 1885, 1974 N45D/T163I/K167R/R195G 1886, 1975 N45D/T183A/T192S/R194G 1887, 1976 N45D/V50A/I119T/K144E 1888, 1977 T19A/N45D/K144E/R195G 1889, 1978 V11E/N45D/T130A/P198T 1890, 1979 V26A/N45D/T163I/T185A 1891, 1980 K23N/N45D/L124S/K167T/R195G 1892, 1981 K23N/N45D/Q73R/T163I 1893, 1982 K28E/N45D/W149R/S158G/P198T 1894, 1983 K28R/N45D/K57E/I98V/R195S 1895, 1984 K28R/N45D/V129D/T163N/R195T 1896, 1985 M41K/D43G/N45D/R64S/R195G 1897, 1986 M41K/D43G/N45D/R64S/S99G 1898, 1987 2006, 2021 N45D/R68L/F173L/D197G/P198S 1899, 1988 N45D/V50A/I148V/R195G/N201D 1900, 1989 M41K/D43G/K44E/N45D/ 1901, 1990 R195G/N201D N45D/V50A/L124S/K144E/ 1902, 1991 L179P/R195G

The number of such non-affinity modified or affinity modified IgSF domains present in a “stacked” immunomodulatory protein construct (whether non-wild type combinations or non-wild type arrangements) is at least 2, 3, 4, or 5 and in some embodiments exactly 2, 3, 4, or 5 IgSF domains (whereby determination of the number of affinity modified IgSF domains disregards any non-specific binding fractional sequences thereof and/or substantially immunologically inactive fractional sequences thereof).

In some embodiments of a stacked immunomodulatory protein provided herein, the number of IgSF domains is at least 2 wherein the number of affinity modified and the number of non-affinity modified IgSF domains is each independently at least: 0, 1, 2, 3, 4, 5, or 6. Thus, the number of affinity modified IgSF domains and the number of non-affinity modified IgSF domains, respectively, (affinity modified IgSF domain: non-affinity modified IgSF domain), can be exactly or at least: 2:0 (affinity modified: wild-type), 0:2, 2:1, 1:2, 2:2, 2:3, 3:2, 2:4, 4:2, 1:1, 1:3, 3:1, 1:4, 4:1, 1:5, or 5:1.

In some embodiments of a stacked immunomodulatory protein, at least two of the non-affinity modified and/or affinity modified IgSF domains are identical IgSF domains.

In some embodiments, a stacked immunomodulatory protein provided herein comprises at least two affinity modified and/or non-affinity modified IgSF domains from a single IgSF member but in a non-wild-type arrangement (alternatively, “permutation”). One illustrative example of a non-wild type arrangement or permutation is an immunomodulatory protein comprising a non-wild-type order of affinity modified and/or non-affinity modified IgSF domain sequences relative to those found in the wild-type PD-L2 whose IgSF domain sequences served as the source of the variant IgSF domains as provided herein. Thus, in one example, the immunomodulatory protein can comprise an IgV proximal and an IgC distal to the transmembrane domain albeit in a non-affinity modified and/or affinity modified form. The presence, in an immunomodulatory protein provided herein, of both non-wild-type combinations and non-wild-type arrangements of non-affinity modified and/or affinity modified IgSF domain is also within the scope of the provided subject matter.

In some embodiments of a stacked immunomodulatory protein, the non-affinity modified and/or affinity modified IgSF domains are non-identical (i.e., different) IgSF domains. Non-identical affinity modified IgSF domains specifically bind, under specific binding conditions, different cognate binding partners and are “non-identical” irrespective of whether or not the wild-type or unmodified IgSF domains from which they are engineered was the same. Thus, for example, a non-wild-type combination of at least two non-identical IgSF domains in an immunomodulatory protein can comprise at least one IgSF domain sequence whose origin is from and unique to one PD-L2, and at least one of a second IgSF domain sequence whose origin is from and unique to another IgSF family member that is not PD-L2, wherein the IgSF domains of the immunomodulatory protein are in non-affinity modified and/or affinity modified form. However, in alternative embodiments, the two non-identical IgSF domains originate from the same IgSF domain sequence but at least one is affinity modified such that they specifically bind to different cognate binding partners.

A plurality of non-affinity modified and/or affinity modified IgSF domains in a stacked immunomodulatory protein polypeptide chain need not be covalently linked directly to one another. In some embodiments, an intervening span of one or more amino acid residues indirectly covalently bonds the non-affinity modified and/or affinity modified IgSF domains to each other. The linkage can be via the N-terminal to C-terminal residues.

In some embodiments, the two or more IgSF domain, including a vIgD of PD-L2 and one or more additional IgSF domain (e.g. second or third variant IgSF domain) from another IgSF family member, are covalently or non-covalently linked. In some embodiments, the two or more IgSF domains are linked directly or indirectly, such as via a linker. In some embodiments, an intervening span of one or more amino acid residues indirectly covalently bonds IgSF domains to each other. The linkage can be via the N-terminal to C-terminal residues. In some embodiments, the linkage can be made via side chains of amino acid residues that are not located at the N-terminus or C-terminus of the IgSF domain(s). Thus, linkages can be made via terminal or internal amino acid residues or combinations thereof.

In some embodiments, the immunomodulatory protein contains at least two IgSF domains, each linked directly or indirectly via a linker. In some embodiments, the immunomodulatory protein contains at least three immunomodulatory proteins, each linked directly or indirectly via a linker. Various configurations are shown in FIGS. 5A and 5B.

In some embodiments, one or more “peptide linkers” link the vIgD of PD-L2 and one or more additional IgSF domain (e.g. second or third variant IgSF domain). In some embodiments, a peptide linker can be a single amino acid residue or greater in length. In some embodiments, the peptide linker has at least one amino acid residue but is no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues in length. In some embodiments, the linker is a flexible linker. In some embodiments, the linker is (in one-letter amino acid code): GGGGS (“4GS”) or multimers of the 4GS linker, such as repeats of 2, 3, 4, or 5 4GS linkers. In some embodiments, the peptide linker is (GGGGS)₂(SEQ ID NO:264) or (GGGGS)₃(SEQ ID NO:263). In some embodiments, the linker also can include a series of alanine residues alone or in addition to another peptide linker (such as a 4GS linker or multimer thereof). In some embodiments, the number of alanine residues in each series is: 2, 3, 4, 5, or 6 alanines. In some embodiments, the linker also can include a series of alanine residues alone or in addition to another peptide linker (such as a 4GS linker or multimer thereof). In some embodiments, the number of alanine residues in each series is: 2, 3, 4, 5, or 6 alanines. In some embodiments, the linker is a rigid linker. For example, the linker is an a-helical linker. In some embodiments, the linker is (in one-letter amino acid code): EAAAK or multimers of the EAAAK linker, such as repeats of 2, 3, 4, or 5 EAAAK linkers, such as set forth in SEQ ID NO: 2030 (1×EAAAK), SEQ ID NO: 2031 (3×EAAAK) or SEQ ID NO: 2032 (5×EAAAK). In some embodiments, the linker can further include amino acids introduced by cloning and/or from a restriction site, for example the linker can include the amino acids GS (in one-letter amino acid code) as introduced by use of the restriction site BAMHI. For example, in some embodiments, the linker (in one-letter amino acid code) is GSGGGGS (SEQ ID NO:1741), GS(G₄S)₃ (SEQ ID NO: 2040), or GS(G₄S)₅ (SEQ ID NO: 2041). In some examples, the linker is a 2×GGGGS followed by three alanines (GGGGSGGGGSAAA; SEQ ID NO: 265). In some cases, various combinations of peptide linkers are used.

In some embodiments, the non-affinity modified and/or affinity modified IgSF domains are linked by “wild-type peptide linkers” inserted at the N-terminus and/or C-terminus of a non-affinity modified and/or affinity modified IgSF domains. These linkers are also called leading sequences (N-terminal to non-affinity modified or affinity modified IgSF domain) or trailing sequences (C-terminal to non-affinity modified or affinity modified IgSF domain), and sequences that exist in the wild-type protein that span immediately outside the structural prediction of the Ig fold of the IgSF. In some embodiments, the “wild-type linker” is an amino acid sequence that exists after the signal sequence, but before in the IgSF domain, such as the defined IgV domain, in the amino acid sequence of the wild-type protein. In some embodiments, the “wild-type” linker is an amino acid sequence that exists immediately after the IgSF domain, such as immediately after the defined IgV domain but before the IgC domain, in the amino acid sequence of the wild-type protein. These linker sequences can contribute to the proper folding and function of the neighboring IgSF domain(s). In some embodiments, there is present a leading peptide linker inserted at the N-terminus of the first IgSF domain and/or a trailing sequence inserted at the C-terminus of the first non-affinity modified and/or affinity modified IgSF domain. In some embodiments, there is present a second leading peptide linker inserted at the N-terminus of the second IgSF domain and/or a second trailing sequence inserted at the C-terminus of the second non-affinity modified and/or affinity modified IgSF domain. When the first and second non-affinity modified and/or affinity modified IgSF domains are derived from the same parental protein and are connected in the same orientation, wild-type peptide linkers between the first and second non-affinity modified and/or affinity modified IgSF domains are not duplicated. For example, when the first trailing wild-type peptide linker and the second leading wild-type peptide linker are the same, the Type II immunomodulatory protein does not comprise either the first trailing wild-type peptide linker or the second leading wild-type peptide linker.

In some embodiments, the Type II immunomodulatory protein comprises a first leading wild-type peptide linker inserted at the N-terminus of the first non-affinity modified and/or affinity modified IgSF domain, wherein the first leading wild-type peptide linker comprises at least 5 (such as at least about any of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more) consecutive amino acids from the intervening sequence in the wild-type protein from which the first non-affinity modified and/or affinity modified IgSF domain is derived between the parental IgSF domain and the immediately preceding domain (such as a signal peptide or an IgSF domain). In some embodiments, the first leading wild-type peptide linker comprises the entire intervening sequence in the wild-type protein from which the first non-affinity modified and/or affinity modified IgSF domain is derived between the parental IgSF domain and the immediately preceding domain (such as a signal peptide or an IgSF domain).

In some embodiments, the Type II immunomodulatory protein further comprises a first trailing wild-type peptide linker inserted at the C-terminus of the first non-affinity modified and/or affinity modified IgSF domain, wherein the first trailing wild-type peptide linker comprises at least 5 (such as at least about any of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more) consecutive amino acids from the intervening sequence in the wild-type protein from which the first non-affinity modified and/or affinity modified IgSF domain is derived between the parental IgSF domain and the immediately following domain (such as an IgSF domain or a transmembrane domain). In some embodiments, the first trailing wild-type peptide linker comprises the entire intervening sequence in the wild-type protein from which the first non-affinity modified and/or affinity modified IgSF domain is derived between the parental IgSF domain and the immediately following domain (such as an IgSF domain or a transmembrane domain).

In some embodiments, the Type II immunomodulatory protein further comprises a second leading wild-type peptide linker inserted at the N-terminus of the second non-affinity modified and/or affinity modified IgSF domain, wherein the second leading wild-type peptide linker comprises at least 5 (such as at least about any of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more) consecutive amino acids from the intervening sequence in the wild-type protein from which the second non-affinity modified and/or affinity modified IgSF domain is derived between the parental IgSF domain and the immediately preceding domain (such as a signal peptide or an IgSF domain). In some embodiments, the second leading wild-type peptide linker comprises the entire intervening sequence in the wild-type protein from which the second non-affinity modified and/or affinity modified IgSF domain is derived between the parental IgSF domain and the immediately preceding domain (such as a signal peptide or an IgSF domain).

In some embodiments, the Type II immunomodulatory protein further comprises a second trailing wild-type peptide linker inserted at the C-terminus of the second non-affinity modified and/or affinity modified IgSF domain, wherein the second trailing wild-type peptide linker comprises at least 5 (such as at least about any of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more) consecutive amino acids from the intervening sequence in the wild-type protein from which the second non-affinity modified and/or affinity modified IgSF domain is derived between the parental IgSF domain and the immediately following domain (such as an IgSF domain or a transmembrane domain). In some embodiments, the second trailing wild-type peptide linker comprises the entire intervening sequence in the wild-type protein from which the second non-affinity modified and/or affinity modified IgSF domain is derived between the parental IgSF domain and the immediately following domain (such as an IgSF domain or a transmembrane domain).

In some embodiments, the two or more IgSF domain, including a vIgD of PD-L2 and one or more additional IgSF domain (e.g. second and/or third variant IgSF domain) from another IgSF family member, are linked or attached to a multimerization domain, such as to an Fc to form an Fc fusion, which, upon expression in a cell can, in some aspects, produce a dimeric multi-domain stack immunomodulatory protein. Thus, also provided are dimeric multi-domain immunomodulatory proteins.

In some embodiments, the variant PD-L2 polypeptide and one or more additional IgSF domain are independently linked, directly or indirectly, to the N- or C-terminus of a multimerization domain, such as an Fc region. In some embodiments, the variant PD-L2 polypeptide and at least one of the one or more additional IgSF domain are linked, directly or indirectly, and one of the variant PD-L2 and one of the one or more additional IgSF domain is also linked, directly or indirectly, to the N- or C-terminus of the multimerization domain, such as an Fc region. In some embodiments, the N- or C-terminus of the multimerization domain, such as an Fc region, is linked to the variant PD-L2 polypeptide or the one or more additional IgSF domain and the other of the N- or C-terminus of the Fc region is linked to the other of the PD-L2 variant or another of the one or more additional IgSF domain. In some embodiments, linkage to the multimerization domain, such as to an Fc, is via a peptide linker, e.g. a peptide linker, such as described above. In some embodiments, linkage between the variant PD-L2 and the one or more additional second IgSF domain is via a peptide linker, e.g. a peptide linker, such as described above. In some embodiments, the vIgD of PD-L2, the one or more additional IgSF domains, and the multimerization domain, such as an Fc domain, can be linked together in any of numerous configurations as depicted in FIGS. 5A and 5B. Exemplary configurations are described in the Examples.

In some embodiments, the stacked immunomodulatory protein is a dimer formed by two immunomodulatory Fc fusion polypeptides. Also provided are nucleic acid molecules encoding any of the stacked immunomodulatory proteins. In some embodiments, the dimeric multi-domain stack immunomodulatory protein can be produced in cells by expression, or in some cases co-expression, of stack immunomodulatory Fc fusion polypeptides, such as described above in accord with generating dimeric Fc fusion proteins.

In some embodiments, the dimeric multi-domain stack immunomodulatory protein is divalent for each Fc region, monovalent for each subunit, or divalent for one subunit and tetravalent for the other.

In some embodiments, the dimeric multi-domain stack immunomodulatory protein is a homodimeric multi-domain stack Fc protein. In some embodiments, the dimeric multi-domain stack immunomodulatory protein comprises a first stack immunomodulatory Fc fusion polypeptide and a second stack immunomodulatory Fc fusion polypeptide in which the first and second polypeptide are the same. In some embodiments, the multi-domain stack molecule contains a first Fc fusion polypeptide containing a variant PD-L2 and a second IgSF domain and a second Fc fusion polypeptide containing the variant PD-L2 and the second IgSF domain. In some embodiments, the multi-domain stack molecule contains a first Fc fusion polypeptide containing a variant PD-L2, a second IgSF domain, and a third IgSF domain and a second Fc fusion polypeptide containing the variant PD-L2, the second IgSF domain, and the third IgSF domain. In some embodiments, the Fc portion of the first and/or second fusion polypeptide can be any Fc as described above. In some embodiments, the Fc portion or region of the first and second fusion polypeptide is the same.

In some embodiments, the multi-domain stack molecule is heterodimeric, comprising two different Fc fusion polypeptides, e.g. a first and a second Fc polypeptide, wherein at least one is an Fc polypeptide containing a is an Fc fusion polypeptide containing at least one variant PD-L2 polypeptide and/or at least one second IgSF domain (e.g. second variant IgSF domain). In some embodiments, the first or second Fc fusion polypeptide further contains a third IgSF domain (e.g. third variant IgSF domain). In some embodiments, the multi-domain stack molecule contains a first Fc fusion polypeptide containing a variant PD-L2 and a second Fc fusion polypeptide containing at a second IgSF domain, in which, in some cases, the first or second Fc fusion polypeptide additionally contains a third IgSF domain. In some embodiments, the multi-domain stack molecule contains a first Fc fusion polypeptide containing a variant PD-L2, a second IgSF domain, and in some cases, a third IgSF domain and a second Fc fusion polypeptide that is not linked to either a variant PD-L2 polypeptide or an additional IgSF domain. In some embodiments, the Fc portion or region of the first and second fusion polypeptide is the same. In some embodiments, the Fc portion or region of the first and second fusion polypeptide is different.

In some embodiments, the multi-domain stack molecule contains a first Fc fusion polypeptide containing 1, 2, 3, 4 or more variant PD-L2 polypeptides and 1, 2, 3, 4 or more additional IgSF domains, wherein the total number of IgSF domains in the first stack Fc fusion polypeptide is greater than 2, 3, 4, 5, 6 or more. In one example of such an embodiment, the second stack Fc polypeptide contains 1, 2, 3, 4 or more additional variant PD-L2 polypeptides and 1, 2, 3, 4 or more second IgSF domains, wherein the total number of IgSF domains in the second stack Fc fusion polypeptide is greater than 2, 3, 4, 5, 6 or more. In another example of such embodiments, the second Fc fusion polypeptide is not linked to either a variant PD-L2 polypeptide or additional IgSF domain.

In some embodiments, the heterodimeric stack molecule contains a first stack immunomodulatory Fc fusion polypeptide and a second stack immunomodulatory Fc fusion polypeptide in which the first and second polypeptide are different. In some embodiments, a heterodimeric stack molecule contains a first Fc polypeptide fusion containing an Fc region and a first variant PD-L2 polypeptide and/or second IgSF domain (e.g. second variant IgSF domain) and a second Fc polypeptide fusion containing an Fc region and the other of the first variant PD-L2 polypeptide or the second IgSF domain. In some embodiments, a heterodimeric stack molecule contains a first Fc polypeptide fusion containing an Fc region and a first variant PD-L2 polypeptide and/or second IgSF domain (e.g. second variant IgSF domain) and a second Fc subunit containing both the first variant PD-L2 polypeptide and second IgSF domain (e.g. second variant IgSF domain) but in a different orientation or configuration from the first Fc region. In some embodiments, the first and/or second Fc fusion polypeptide also contains a third IgSF domain (e.g. third variant IgSF domain).

In some embodiments, the Fc domain of one or both of the first and second stacked immunomodulatory Fc fusion polypeptide comprises a modification (e.g. substitution) such that the interface of the Fc molecule is modified to facilitate and/or promote heterodimerization. In some embodiments, modifications include introduction of a protuberance (knob) into a first Fc polypeptide and a cavity (hole) into a second Fc polypeptide such that the protuberance is positionable in the cavity to promote complexing of the first and second Fc-containing polypeptides. Amino acids targeted for replacement and/or modification to create protuberances or cavities in a polypeptide are typically interface amino acids that interact or contact with one or more amino acids in the interface of a second polypeptide.

In some embodiments, a sequence of amino acids is added preceding the Fc sequence for constructs in which the Fc sequence was the N-terminal portion of the sequence. In some cases, the sequence of amino acids HMSSVSAQ (SEQ ID NO:1190) is added immediately preceding the Fc sequence for constructs in which the Fc sequence was the N-terminal portion of the sequence. In some embodiments, a heterodimeric stack molecule contains a first Fc polypeptide fusion containing an Fc region (knob) and a first variant polypeptide and/or second IgSF domain (e.g. second variant IgSF domain) and a second Fc polypeptide fusion containing an Fc region (hole) and a stuffer sequence HMSSVSAQ (SEQ ID NO:1190) was added immediately preceding both Fc regions of the first and second Fc polypeptide fusion.

In some embodiments, a first polypeptide that is modified to contain protuberance (hole) amino acids include replacement of a native or original amino acid with an amino acid that has at least one side chain which projects from the interface of the first polypeptide and is therefore positionable in a compensatory cavity (hole) in an adjacent interface of a second polypeptide. Most often, the replacement amino acid is one which has a larger side chain volume than the original amino acid residue. One of skill in the art knows how to determine and/or assess the properties of amino acid residues to identify those that are ideal replacement amino acids to create a protuberance. In some embodiments, the replacement residues for the formation of a protuberance are naturally occurring amino acid residues and include, for example, arginine (R), phenylalanine (F), tyrosine (Y), or tryptophan (W). In some examples, the original residue identified for replacement is an amino acid residue that has a small side chain such as, for example, alanine, asparagine, aspartic acid, glycine, serine, threonine, or valine.

In some embodiments, a second polypeptide that is modified to contain a cavity (hole) is one that includes replacement of a native or original amino acid with an amino acid that has at least one side chain that is recessed from the interface of the second polypeptide and thus is able to accommodate a corresponding protuberance from the interface of a first polypeptide. Most often, the replacement amino acid is one which has a smaller side chain volume than the original amino acid residue. One of skill in the art knows how to determine and/or assess the properties of amino acid residues to identify those that are ideal replacement residues for the formation of a cavity. Generally, the replacement residues for the formation of a cavity are naturally occurring amino acids and include, for example, alanine (A), serine (S), threonine (T) and valine (V). In some examples, the original amino acid identified for replacement is an amino acid that has a large side chain such as, for example, tyrosine, arginine, phenylalanine, or tryptophan.

The CH3 interface of human IgG1, for example, involves sixteen residues on each domain located on four anti-parallel β-strands which buries 1090 Å2 from each surface (see e.g., Deisenhofer et al. (1981) Biochemistry, 20:2361-2370; Miller et al., (1990) J Mol. Biol., 216, 965-973; Ridgway et al., (1996) Prot. Engin., 9: 617-621; U.S. Pat. No. 5,731,168). Modifications of a CH3 domain to create protuberances or cavities are described, for example, in U.S. Pat. No. 5,731,168; International Patent Applications WO98/50431 and WO 2005/063816; and Ridgway et al., (1996) Prot. Engin., 9: 617-621. In some examples, modifications of a CH3 domain to create protuberances or cavities are typically targeted to residues located on the two central anti-parallel β-strands. The aim is to minimize the risk that the protuberances which are created can be accommodated by protruding into the surrounding solvent rather than being accommodated by a compensatory cavity in the partner CH3 domain.

In some embodiments, the heterodimeric molecule contains a T366W mutation in the CH3 domain of the “knobs chain” and T366S, L368A, Y407V mutations in the CH3 domain of the “hole chain”. In some cases, an additional interchain disulfide bridge between the CH3 domains can also be used (Merchant, A. M., et al., Nature Biotech. 16 (1998) 677-681) e.g. by introducing a Y349C mutation into the CH3 domain of the “knobs” or “hole” chain and a E356C mutation or a S354C mutation into the CH3 domain of the other chain. In some embodiments, the heterodimeric molecule contains S354C, T366W mutations in one of the two CH3 domains and Y349C, T366S, L368A, Y407V mutations in the other of the two CH3 domains. In some embodiments, the heterodimeric molecule comprises E356C, T366W mutations in one of the two CH3 domains and Y349C, T366S, L368A, Y407V mutations in the other of the two CH3 domains. In some embodiments, the heterodimeric molecule comprises Y349C, T366W mutations in one of the two CH3 domains and E356C, T366S, L368A, Y407V mutations in the other of the two CH3 domains. In some embodiments, the heterodimeric molecule comprises Y349C, T366W mutations in one of the two CH3 domains and S354C, T366S, L368A, Y407V mutations in the other of the two CH3 domains. Examples of other knobs-in-holes technologies are known in the art, e.g. as described by EP 1 870 459 A1.

In some embodiments, the Fc regions of the heterodimeric molecule additionally can contain one or more other Fc mutation, such as any described above. In some embodiments, the heterodimer molecule contains an Fc region with a mutation that reduces effector function.

In some embodiments, an Fc variant containing CH3 protuberance (knob) or cavity (hole) modifications can be joined to a stacked immunomodulatory polypeptide anywhere, but typically via its N- or C-terminus, to the N- or C-terminus of a first and/or second stacked immunomodulatory polypeptide, such as to form a fusion polypeptide. The linkage can be direct or indirect via a linker. Typically, a knob and hole molecule is generated by co-expression of a first stacked immunomodulatory polypeptide linked to an Fc variant containing CH3 protuberance modification(s) with a second stacked immunomodulatory polypeptide linked to an Fc variant containing CH3 cavity modification(s).

There is provided herein a homodimeric multi-domain stack molecule produced from a stack immunomodulatory Fc fusion polypeptide containing an IgSF domain, e.g. IgV domain, of a variant CD155 polypeptide and a second IgSF domain, e.g. IgV, of a variant PD-L2 polypeptide. In some embodiments, the first and second immunomodulatory Fc fusion polypeptide of the multi-domain stack molecule has the sequence set forth in any of SEQ ID NOS: 1191, 1192, 1193, 1194, 1195, or 1196 or a sequence of amino acids that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOS: 1191, 1192, 1193, 1194, 1195, or 1196 and contains the one more amino acid modifications in the variant PD-L2 and/or CD155 IgSF domain. In some embodiments, the resulting multi-domain stack molecules bind to both TIGIT and PD-1. In some aspects, the binding to TIGIT is to the same or similar degree or, in some cases, is increased, compared to the binding to TIGIT of the corresponding IgSF domain of unmodified or wild-type CD155. In some aspects, the binding to PD-1 is to the same or similar degree or, in some cases, is increased, compared to the binding to PD-1 of the corresponding IgSF domain of unmodified or wild-type PD-L1. In some embodiments, the binding to TIGIT or PD-1 is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of the binding to TIGIT or PD-1 of the non-stacked form of the variant CD155 IgSF-Fc. In some embodiments, the binding to TIGIT is at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the binding to TIGIT of the non-stacked form of the variant CD155 IgSF-Fc. In some embodiments, the resulting multi-domain stack molecule increases T cell immune responses compared to the non-stack variant PD-L2 IgSF-Fc and/or variant CD155-IgSF-Fc, such as determined in a reporter assay. In some embodiments, the increase is greater than 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2.0-fold, 3.0-fold, 4.0-fold, 5.0-fold or more.

There is provided herein a heterodimeric multi-domain stack molecule produced from a stack immunomodulatory Fc fusion polypeptide containing an IgSF domain, e.g. IgV domain, of a variant CD155 polypeptide and a second IgSF domain, e.g. IgV, of a variant PD-L2 polypeptide. In some embodiments, one of the first and second immunomodulatory Fc fusion polypeptide of the multi-domain stack molecule comprises a knob molecule that has the sequence set forth in any of SEQ ID NOS: 1197, 1198, 1199, 1200, 1201, or 1203, or a sequence of amino acids that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOS: 1197, 1198, 1199, 1200, 1201, or 1203, and contains the one more amino acid modifications in the variant PD-L2 and/or CD155 IgSF domain. In some embodiments, the other of the first and second immunomodulatory Fc fusion polypeptide of the multi-domain stack molecule comprises a hole molecule that has the sequence set forth in any of SEQ ID NOS: 1188, 1202, or 1204, or a sequence of amino acids that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOS: 1188, 1202, or 1204, and contains the one more amino acid modifications in the variant PD-L2 and/or CD155 IgSF domain. In some embodiments, the resulting multi-domain stack molecules bind to both TIGIT and PD-1. In some embodiments, the knob and hole molecules are expressed in various combinations and are generated by co-expression of a first stacked immunomodulatory polypeptide linked to an Fc variant containing CH3 protuberance modification(s) with a second stacked immunomodulatory polypeptide linked to an Fc variant containing CH3 cavity modification(s). For example, the first and second immunomodulatory Fc fusion polypeptide of the multi-domain stack molecule comprises a knob and hole molecule that has the pair of sequences set forth in any of SEQ ID NOS:1197+1188, 1198+1188, 1199+1188, 1200+1188, 1201+1202, 1203+1204, 1199+1204, 1199+1202, 1200+1202, 1200+1204, or a sequence of amino acids that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of the pairs of SEQ ID NOS: 1197+1188, 1198+1188, 1199+1188, 1200+1188, 1201+1202, 1203+1204, 1199+1204, 1199+1202, 1200+1202, 1200+1204, and contains the one more amino acid modifications in the variant PD-L2 and/or CD155 IgSF domain. In some cases, the knob or hole molecule includes a N-terminal HMSSVSAQ set forth in SEQ ID NO:1190. In some aspects, the binding to TIGIT is to the same or similar degree or, in some cases, is increased, compared to the binding to TIGIT of the corresponding IgSF domain of unmodified or wild-type CD155. In some aspects, the binding to PD-1 is to the same or similar degree or, in some cases, is increased, compared to the binding to PD-1 of the corresponding IgSF domain of unmodified or wild-type PD-L2. In some embodiments, the binding to TIGIT or PD-1 is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of the binding to TIGIT or PD-1 of the non-stacked form of the variant CD155 IgSF-Fc. In some embodiments, the binding to TIGIT is at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the binding to TIGIT of the non-stacked form of the variant CD155 IgSF-Fc. In some embodiments, the resulting multi-domain stack molecule increases T cell immune responses compared to the non-stack variant PD-L2 IgSF-Fc and/or variant CD155-IgSF-Fc, such as determined in a reporter assay. In some embodiments, the increase is greater than 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2.0-fold, 3.0-fold, 4.0-fold, 5.0-fold or more.

C. Conjugates and Fusions of Variant Polypeptides and Immunomodulatory Proteins

In some embodiments, the variant polypeptides provided herein, which are immunomodulatory proteins comprising variants of an Ig domain of the IgSF family (vIgD), can be conjugated with or fused with a moiety, such as an effector moiety, such as another protein, directly or indirectly, to form a conjugate (“IgSF conjugate”). In some embodiments, the attachment can be covalent or non-covalent, e.g., via a biotin-streptavidin non-covalent interaction. In some embodiments, the moiety can be a targeting moiety, a small molecule drug (non-polypeptide drug of less than 500 daltons molar mass), a toxin, a cytostatic agent, a cytotoxic agent, an immunosuppressive agent, a radioactive agent suitable for diagnostic purposes, a radioactive metal ion for therapeutic purposes, a prodrug-activating enzyme, an agent that increases biological half-life, or a diagnostic or detectable agent.

In some embodiments the effector moiety is a therapeutic agent, such as a cancer therapeutic agent, which is either cytotoxic, cytostatic or otherwise provides some therapeutic benefit. In some embodiments, the effector moiety is a targeting moiety or agent, such as an agent that targets a cell surface antigen, e.g., an antigen on the surface of a tumor cell. In some embodiments, the effector moiety is a label, which can generate a detectable signal, either directly or indirectly. In some embodiments, the effector moiety is a toxin. In some embodiments, the effector moiety is a protein, peptide, nucleic acid, small molecule or nanoparticle.

In some embodiments, 1, 2, 3, 4, 5 or more effector moieties, which can be the same or different, are conjugated, linked or fused to the variant polypeptide or protein to form an IgSF conjugate. In some embodiments, such effector moieties can be attached to the variant polypeptide or immunomodulatory protein using various molecular biological or chemical conjugation and linkage methods known in the art and described below. In some embodiments, linkers such as peptide linkers, cleavable linkers, non-cleavable linkers or linkers that aid in the conjugation reaction, can be used to link or conjugate the effector moieties to the variant polypeptide or immunomodulatory protein.

In some embodiments, the IgSF conjugate comprises the following components: (protein or polypeptide), (L)_(q) and (effector moiety)_(m), wherein the protein or polypeptide is any of the described variant polypeptides or immunomodulatory proteins capable of binding one or more cognate counter structure ligands as described; L is a linker for linking the protein or polypeptide to the moiety; m is at least 1; q is 0 or more; and the resulting IgSF conjugate binds to the one or more counter structure ligands. In particular embodiments, m is 1 to 4 and q is 0 to 8.

In some embodiments, there is provided an IgSF conjugate comprising a variant polypeptide or immunomodulatory protein provided herein conjugated with a targeting agent that binds to a cell surface molecule, for example, for targeted delivery of the variant polypeptide or immunomodulatory protein to a specific cell. In some embodiments, the targeting agent is a molecule(s) that has the ability to localize and bind to a molecule present on a normal cell/tissue and/or tumor cell/tumor in a subject. In other words, IgSF conjugates comprising a targeting agent can bind to a ligand (directly or indirectly), which is present on a cell, such as a tumor cell. The targeting agents of the invention contemplated for use include antibodies, polypeptides, peptides, aptamers, other ligands, or any combination thereof, that can bind a component of a target cell or molecule.

In some embodiments, the targeting agent binds a tumor cell(s) or can bind in the vicinity of a tumor cell(s) (e.g., tumor vasculature or tumor microenvironment) following administration to the subject. The targeting agent may bind to a receptor or ligand on the surface of the cancer cell. In another aspect of the invention, a targeting agent is selected which is specific for a noncancerous cells or tissue. For example, a targeting agent can be specific for a molecule present normally on a particular cell or tissue. Furthermore, in some embodiments, the same molecule can be present on normal and cancer cells. Various cellular components and molecules are known. For example, if a targeting agent is specific for EGFR, the resulting IgSF conjugate can target cancer cells expressing EGFR as well as normal skin epidermal cells expressing EGFR. Therefore, in some embodiments, an IgSF conjugate of the invention can operate by two separate mechanisms (targeting cancer and non-cancer cells).

In various aspects of the invention disclosed herein an IgSF conjugate of the invention comprises a targeting agent which can bind/target a cellular component, such as a tumor antigen, a bacterial antigen, a viral antigen, a mycoplasm antigen, a fungal antigen, a prion antigen, an antigen from a parasite. In some aspects, a cellular component, antigen or molecule can each be used to mean, a desired target for a targeting agent. For example, in various embodiments, a targeting agent is specific for or binds to a component, which includes but is not limited to, epidermal growth factor receptor (EGFR, ErbB-1, HER1), ErbB-2 (HER2/neu), ErbB-3/HER3, ErbB-4/HER4, EGFR ligand family; insulin-like growth factor receptor (IGFR) family, IGF-binding proteins (IGFBPs), IGFR ligand family; platelet derived growth factor receptor (PDGFR) family, PDGFR ligand family; fibroblast growth factor receptor (FGFR) family, FGFR ligand family, vascular endothelial growth factor receptor (VEGFR) family, VEGF family; HGF receptor family; TRK receptor family; ephrin (EPH) receptor family; AXL receptor family; leukocyte tyrosine kinase (LTK) receptor family; TIE receptor family, angiopoietin 1,2; receptor tyrosine kinase-like orphan receptor (ROR) receptor family, e.g. ROR1; CD171 (L1CAM); B7-H6 (NCR3LG1); PD-L2, tumor glycosylation antigen, e.g. sTn or Tn, such as sTn Ag of MUC1; LHR (LHCGR); phosphatidylserine, discoidin domain receptor (DDR) family; RET receptor family; KLG receptor family; RYK receptor family; MuSK receptor family; Transforming growth factor-α (TGF-α) receptors, TGF-β; Cytokine receptors, Class I (hematopoietin family) and Class II (interferon/IL-10 family) receptors, tumor necrosis factor (TNF) receptor superfamily (TNFRSF), death receptor family; cancer-testis (CT) antigens, lineage-specific antigens, differentiation antigens, alpha-actinin-4, ARTC1, breakpoint cluster region-Abelson (Bcr-abl) fusion products, B-RAF, caspase-5 (CASP-5), caspase-8 (CASP-8), β-catenin (CTNNB1), cell division cycle 27 (CDC27), cyclin-dependent kinase 4 (CDK4), CDKN2A, COA-I, dek-can fusion protein, EFTUD-2, Elongation factor 2 (ELF2), Ets variant gene 6/acute myeloid leukemia 1 gene ETS (ETC6-AML1) fusion protein, fibronectin (FN), e.g. the extradomain A (EDA) of fibronectin, GPNMB, low density lipid receptor/GDP-L fucose: β-D-galactose 2-a-L-fucosyltransferase (LDLR/FUT) fusion protein, HLA-A2. arginine to isoleucine exchange at residue 170 of the α-helix of the α2-domain in the HLA-A2gene (HLA-A*201-R170I), HLA-Al 1, heat shock protein 70-2 mutated (HSP70-2M), K1AA0205, MART2, melanoma ubiquitous mutated 1, 2, 3 (MUM-I, 2, 3), prostatic acid phosphatase (PAP), neo-PAP, Myosin class I, NFYC, OGT, OS-9, pml-RARα fusion protein, PRDX5, PTPRK, K-ras (KRAS2), N-ras (NRAS), HRAS, RBAF600, SIRT2, SNRPD1, SYT-SSX1 or -SSX2 fusion protein, Triosephosphate Isomerase, BAGE, BAGK-1, BAGE-2,3,4,5, GAGE-1,2,3,4,5,6,7,8, GnT-V (aberrant N-acetyl glucosaminyl transferase V, MGAT5), HERV-K-MEL, KK-LC, KM-HN-I, LAGE, LAGE-I, CTL-recognized antigen on melanoma (CAMEL), MAGE-A1 (MAGE-I), MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A8, MAGE-A9, MAGE-AlO, MAGE-AI1, MAGE-A12, MAGE-3, MAGE-B1, MAGE-B2, MAGE-B5, MAGE-B6, MAGE-Cl, MAGE-C2, mucin 1 (MUC1), MART-1/Melan-A (MLANA), gplOO, gplOO/Pmell7 (SILV), tyrosinase (TYR), TRP-I, HAGE, NA-88, NY-ESO-I, NY-ESO-1/LAGE-2, SAGE, Spl7, SSX-1,2,3,4, TRP2-INT2, carcino-embryonic antigen (CEA), Kallikrein 4, mammaglobin-A, OA1, prostate specific antigen (PSA), TRP-1/gp75, TRP-2, adipophilin, interferon inducible protein absent in melanoma 2 (AIM-2), BING-4, CPSF, cyclin Dl, epithelial cell adhesion molecule (Ep-CAM), EphA3, fibroblast growth factor-5 (FGF-5), glycoprotein 250 (gp250), EGFR (ERBB1), HER-2/neu (ERBB2), interleukin 13 receptor α2 chain (IL13Rα2), IL-6 receptor, intestinal carboxyl esterase (iCE), alpha-feto protein (AFP), M-CSF, mdm-2, MUCl, p53 (TP53), PBF, PRAME, PSMA, RAGE-I, RNF43, RU2AS, SOXlO, STEAPl, survivin (BIRC5), human telomerase reverse transcriptase (hTERT), telomerase, Wilms' tumor gene (WT1), SYCP1, BRDT, SPANX, XAGE, ADAM2, PAGE-5, LIPl, CTAGE-I, CSAGE, MMAl, CAGE, BORIS, HOM-TES-85, AF15q14, HCA661, LDHC, MORC, SGY-I, SPOl 1, TPX1, NY-SAR-35, FTHL17, NXF2, TDRD1, TEX15, FATE, TPTE, immunoglobulin idiotypes, Bence-Jones protein, estrogen receptors (ER), androgen receptors (AR), CD40, CD30, CD20, CD 19, CD33, cancer antigen 72-4 (CA 72-4), cancer antigen 15-3 (CA 15-3), cancer antigen 27-29 (CA 27-29), cancer antigen 125 (CA 125), cancer antigen 19-9 (CA 19-9), β-human chorionic gonadotropin, β-2 microglobulin, squamous cell carcinoma antigen, neuron-specific enolase, heat shock protein gp96, GM2, sargramostim, CTLA-4, 707 alanine proline (707-AP), adenocarcinoma antigen recognized by T cells 4 (ART-4), carcinoembryogenic antigen peptide-1 (CAP-I), calcium-activated chloride channel-2 (CLCA2), cyclophilin B (Cyp-B), human signet ring tumor-2 (HST-2), Human papilloma virus (HPV) proteins (HPV-E6, HPV-E7, major or minor capsid antigens, others), Epstein-Barr virus (EBV) proteins (EBV latent membrane proteins—LMP1, LMP2; others), Hepatitis B or C virus proteins, and HIV proteins.

In some embodiments, an IgSF conjugate, through its targeting agent, will bind a cellular component of a tumor cell, tumor vasculature or tumor microenvironment, thereby promoting killing of targeted cells via modulation of the immune response, (e.g., by activation of co-stimulatory molecules or inhibition of negative regulatory molecules of immune cell activation), inhibition of survival signals (e.g., growth factor or cytokine or hormone receptor antagonists), activation of death signals, and/or immune-mediated cytotoxicity, such as through antibody dependent cellular cytotoxicity. Such IgSF conjugates can function through several mechanisms to prevent, reduce or eliminate tumor cells, such as to facilitate delivery of conjugated effector moieties to the tumor target, such as through receptor-mediated endocytosis of the IgSF conjugate; or such conjugates can recruit, bind, and/or activate immune cells (e.g. NK cells, monocytes/macrophages, dendritic cells, T cells, B cells). Moreover, in some instances one or more of the foregoing pathways may operate upon administration of one or more IgSF conjugates of the invention.

In some embodiments, an IgSF conjugate, through its targeting agent, will be localized to, such as bind to, a cellular component of a tumor cell, tumor vasculature or tumor microenvironment, thereby modulating cells of the immune response in the vicinity of the tumor. In some embodiments, the targeting agent facilitates delivery of the conjugated IgSF (e.g. vIgD) to the tumor target, such as to interact with its cognate binding partner to alter signaling of immune cells (e.g. NK cells, monocytes/macrophages, dendritic cells, T cells, B cells) bearing the cognate binding partner. In some embodiments, localized delivery mediates an antagonizing or blocking activity of the PD-1 inhibitory receptor. In some embodiments, localized delivery agonizes the PD-1 inhibitory receptor, which, in some cases, can occur where there is proximal clustering of an activating receptor.

In some embodiments, the targeting agent is an immunoglobulin. As used herein, the term “immunoglobulin” includes natural or artificial mono- or polyvalent antibodies including, but not limited to, polyclonal, monoclonal, multispecific, human, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab′) fragments, fragments produced by a Fab expression library, single chain Fv (scFv); anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the invention), and epitope-binding fragments of any of the above. The term “antibody,” as used herein, refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, e.g., molecules that contain an antigen binding site that immunospecifically binds an antigen. The immunoglobulin molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass of immunoglobulin molecule.

In some embodiments, an IgSF conjugate, through its antibody targeting moiety, will bind a cellular component of a tumor cell, tumor vasculature or tumor microenvironment, thereby promoting apoptosis of targeted cells via modulation of the immune response, (e.g., by activation of co-stimulatory molecules or inhibition of negative regulatory molecules of immune cell activation), inhibition of survival signals (e.g., growth factor or cytokine or hormone receptor antagonists), activation of death signals, and/or immune-mediated cytotoxicity, such as through antibody dependent cellular cytotoxicity. Such IgSF conjugates can function through several mechanisms to prevent, reduce or eliminate tumor cells, such as to facilitate delivery of conjugated effector moieties to the tumor target, such as through receptor-mediated endocytosis of the IgSF conjugate; or such conjugates can recruit, bind, and/or activate immune cells (e.g. NK cells, monocytes/macrophages, dendritic cells, T cells, B cells).

In some embodiments, an IgSF conjugate, through its antibody targeting moiety, will bind a cellular component of a tumor cell, tumor vasculature or tumor microenvironment, thereby modulating the immune response (e.g., by activation of co-stimulatory molecules or inhibition of negative regulatory molecules of immune cell activation). In some embodiments, such conjugates can recognize, bind, and/or modulate (e.g. inhibit or activate) immune cells (e.g. NK cells, monocytes/macrophages, dendritic cells, T cells, B cells).

Antibody targeting moieties of the invention include antibody fragments that include, but are not limited to, Fab, Fab′ and F(ab′)2, Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments comprising either a VL or VH domain. Antigen-binding antibody fragments, including single-chain antibodies, may comprise the variable region(s) alone or in combination with the entirety or a portion of the following: hinge region, CH1, CH2, and CH3 domains. Also included in the invention are antigen-binding fragments also comprising any combination of variable region(s) with a hinge region, CH1, CH2, and CH3 domains. Also included in the invention are Fc fragments, antigen-Fc fusion proteins, and Fc-targeting moiety conjugates or fusion products (Fc-peptide, Fc-aptamer). The antibody targeting moieties of the invention may be from any animal origin including birds and mammals. In one aspect, the antibody targeting moieties are human, murine (e.g., mouse and rat), donkey, sheep, rabbit, goat, guinea pig, camel, horse, or chicken. Further, such antibodies may be humanized versions of animal antibodies. The antibody targeting moieties of the invention may be monospecific, bispecific, trispecific, or of greater multispecificity.

In various embodiments, an antibody/targeting moiety recruits, binds, and/or activates immune cells (e.g. NK cells, monocytes/macrophages, dendritic cells) via interactions between Fc (in antibodies) and Fc receptors (on immune cells) and via the conjugated variant polypeptides or immunomodulatory proteins provided herein. In some embodiments, an antibody/targeting moiety recognizes or binds a tumor agent via and localizes to the tumor cell the conjugated variant polypeptides or immunomodulatory proteins provided herein to facilitate modulation of immune cells in the vicinity of the tumor.

Examples of antibodies which can be incorporated into IgSF conjugates include but are not limited to antibodies such as Cetuximab (IMC-C225; Erbitux®), Trastuzumab (Herceptin®), Rituximab (Rituxan®; MabThera®), Bevacizumab (Avastin®), Alemtuzumab (Campath®; Campath-1H®; Mabcampath®), Panitumumab (ABX-EGF; Vectibix®), Ranibizumab (Lucentis®), Ibritumomab, Ibritumomab tiuxetan, (Zevalin®), Tositumomab, Iodine I 131 Tositumomab (BEXXAR®), Catumaxomab (Removab®), Gemtuzumab, Gemtuzumab ozogamicine (Mylotarg®), Abatacept (CTLA4-Ig; Orencia®), Belatacept (L104EA29YIg; LEA29Y; LEA), Ipilimumab (MDX-010; MDX-101), Tremelimumab (ticilimumab; CP-675,206), PRS-010, PRS-050, Aflibercept (VEGF Trap, AVE005), Volociximab (M200), F200, MORAb-009, SS1P (CAT-5001), Cixutumumab (IMC-A12), Matuzumab (EMD72000), Nimotuzumab (h-R3), Zalutumumab (HuMax-EGFR), Necitumumab IMC-11F8, mAb806/ch806, Sym004, mAb-425, Panorex @ (17-1A) (murine monoclonal antibody); Panorex @ (17-1A) (chimeric murine monoclonal antibody); IDEC-Y2B8 (murine, anti-CD2O MAb); BEC2 (anti-idiotypic MAb, mimics the GD epitope) (with BCG); Oncolym (Lym-1 monoclonal antibody); SMART MI95 Ab, humanized 13′ I LYM-I (Oncolym), Ovarex (B43.13, anti-idiotypic mouse MAb); MDX-210 (humanized anti-HER-2 bispecific antibody); 3622W94 MAb that binds to EGP40 (17-1A) pancarcinoma antigen on adenocarcinomas; Anti-VEGF, Zenapax (SMART Anti-Tac (IL-2 receptor); SMART MI95 Ab, humanized Ab, humanized); MDX-210 (humanized anti-HER-2 bispecific antibody); MDX-447 (humanized anti-EGF receptor bispecific antibody); NovoMAb-G2 (pancarcinoma specific Ab); TNT (chimeric MAb to histone antigens); TNT (chimeric MAb to histone antigens); Gliomab-H (Monoclon s-Humanized Abs); GNI-250 Mab; EMD-72000 (chimeric-EGF antagonist); LymphoCide (humanized LL2 antibody); and MDX-260 bispecific, targets GD-2, ANA Ab, SMART IDlO Ab, SMART ABL 364 Ab or ImmuRAIT-CEA. As illustrated by the forgoing list, it is conventional to make antibodies to a particular target epitope.

In some embodiments, the antibody targeting moiety is a full length antibody, or antigen-binding fragment thereof, containing an Fc domain. In some embodiments, the variant polypeptide or immunomodulatory protein is conjugated to the Fc portion of the antibody targeting moiety, such as by conjugation to the N-terminus of the Fc portion of the antibody.

In some embodiments, the vIgD is linked, directly or indirectly, to the N- or C-terminus of the light and/or heavy chain of the antibody. In some embodiments, linkage can be via a peptide linker, such as any described above. Various configurations can be constructed. FIG. 7A-7C depict exemplary configurations. In some embodiments, the antibody conjugate can be produced by co-expression of the heavy and light chain of the antibody in a cell.

In one aspect of the invention, the targeting agent is an aptamer molecule. For example, in some embodiments, the aptamer is comprised of nucleic acids that function as a targeting agent. In various embodiments, an IgSF conjugate of the invention comprises an aptamer that is specific for a molecule on a tumor cell, tumor vasculature, and/or a tumor microenvironment. In some embodiments, the aptamer itself can comprise a biologically active sequence, in addition to the targeting module (sequence), wherein the biologically active sequence can induce an immune response to the target cell. In other words, such an aptamer molecule is a dual use agent. In some embodiments, an IgSF conjugate of the invention comprises conjugation of an aptamer to an antibody, wherein the aptamer and the antibody are specific for binding to separate molecules on a tumor cell, tumor vasculature, tumor microenvironment, and/or immune cells.

The term “aptamer” includes DNA, RNA or peptides that are selected based on specific binding properties to a particular molecule. For example, an aptamer(s) can be selected for binding a particular gene or gene product in a tumor cell, tumor vasculature, tumor microenvironment, and/or an immune cell, as disclosed herein, where selection is made by methods known in the art and familiar to one of skill in the art.

In some aspects of the invention the targeting agent is a peptide. For example, the variant polypeptides or immunomodulatory proteins provided herein can be conjugated to a peptide which can bind with a component of a cancer or tumor cells. Therefore, such IgSF conjugates of the invention comprise peptide targeting agents which binds to a cellular component of a tumor cell, tumor vasculature, and/or a component of a tumor microenvironment. In some embodiments, targeting agent peptides can be an antagonist or agonist of an integrin. Integrins, which comprise an alpha and a beta subunit, include numerous types well known to a skilled artisan.

In one embodiment, the targeting agent is Vvβ3. Integrin Vvβ3 is expressed on a variety of cells and has been shown to mediate several biologically relevant processes, including adhesion of osteoclasts to bone matrix, migration of vascular smooth muscle cells, and angiogenesis. Suitable targeting molecules for integrins include RGD peptides or peptidomimetics as well as non-RGD peptides or peptidomimetics (see, e.g., U.S. Pat. Nos. 5,767,071 and 5,780,426) for other integrins such as V4.βi (VLA-4), V4-P7 (see, e.g., U.S. Pat. No. 6,365,619; Chang et al, Bioorganic & Medicinal Chem Lett, 12:159-163 (2002); Lin et al., Bioorganic & Medicinal Chem Lett, 12:133-136 (2002)), and the like.

In some embodiments, there is provided an IgSF conjugate comprising a variant polypeptide or immunomodulatory protein provided herein conjugated with a therapeutic agent. In some embodiments, the therapeutic agent includes, for example, daunomycin, doxorubicin, methotrexate, and vindesine (Rowland et al., Cancer Immunol. Immunother. 21:183-187, 1986). In some embodiments, the therapeutic agent has an intracellular activity. In some embodiments, the IgSF conjugate is internalized and the therapeutic agent is a cytotoxin that blocks the protein synthesis of the cell, therein leading to cell death. In some embodiments, the therapeutic agent is a cytotoxin comprising a polypeptide having ribosome-inactivating activity including, for example, gelonin, bouganin, saporin, ricin, ricin A chain, bryodin, diphtheria toxin, restrictocin, Pseudomonas exotoxin A and variants thereof. In some embodiments, where the therapeutic agent is a cytotoxin comprising a polypeptide having a ribosome-inactivating activity, the IgSF conjugate must be internalized upon binding to the target cell in order for the protein to be cytotoxic to the cells.

In some embodiments, there is provided an IgSF conjugate comprising a variant polypeptide or immunomodulatory protein provided herein conjugated with a toxin. In some embodiments, the toxin includes, for example, bacterial toxins such as diphtheria toxin, plant toxins such as ricin, small molecule toxins such as geldanamycin (Mandler et al., J. Nat. Cancer Inst. 92(19):1573-1581 (2000); Mandler et al., Bioorganic & Med. Chem. Letters 10:1025-1028 (2000); Mandler et al., Bioconjugate Chem. 13:786-791 (2002)), maytansinoids (EP 1391213; Liu et al., Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996)), and calicheamicin (Lode et al., Cancer Res. 58:2928 (1998); Hinman et al., Cancer Res. 53:3336-3342 (1993)). The toxins may exert their cytotoxic and cytostatic effects by mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition.

In some embodiments, there is provided an IgSF conjugate comprising a variant polypeptide or immunomodulatory protein provided herein conjugated with a label, which can generate a detectable signal, indirectly or directly. These IgSF conjugates can be used for research or diagnostic applications, such as for the in vivo detection of cancer. The label is preferably capable of producing, either directly or indirectly, a detectable signal. For example, the label may be radio-opaque or a radioisotope, such as 3H, 14C, 32P, 35S, 123I, 125I, 131I; a fluorescent (fluorophore) or chemiluminescent (chromophore) compound, such as fluorescein isothiocyanate, rhodamine or luciferin; an enzyme, such as alkaline phosphatase, β-galactosidase or horseradish peroxidase; an imaging agent; or a metal ion. In some embodiments, the label is a radioactive atom for scintigraphic studies, for example 99Tc or 123I, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as zirconium-89, iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron. Zirconium-89 may be complexed to various metal chelating agents and conjugated to antibodies, e.g., for PET imaging (WO 2011/056983). In some embodiments, the IgSF conjugate is detectable indirectly. For example, a secondary antibody that is specific for the IgSF conjugate and contains a detectable label can be used to detect the IgSF conjugate.

The IgSF conjugates may be prepared using any methods known in the art. See, e.g., WO 2009/067800, WO 2011/133886, and U.S. Patent Application Publication No. 2014322129, incorporated by reference herein in their entirety.

The variant polypeptides or immunomodulatory proteins of an IgSF conjugate may be “attached to” the effector moiety by any means by which the variant polypeptides or immunomodulatory proteins can be associated with, or linked to, the effector moiety. For example, the variant polypeptides or immunomodulatory proteins of an IgSF conjugate may be attached to the effector moiety by chemical or recombinant means. Chemical means for preparing fusions or conjugates are known in the art and can be used to prepare the IgSF conjugate. The method used to conjugate the variant polypeptides or immunomodulatory proteins and effector moiety must be capable of joining the variant polypeptides or immunomodulatory proteins with the effector moiety without interfering with the ability of the variant polypeptides or immunomodulatory proteins to bind to their one or more counter structure ligands.

The variant polypeptides or immunomodulatory proteins of an IgSF conjugate may be linked indirectly to the effector moiety. For example, the variant polypeptides or immunomodulatory proteins of an IgSF conjugate may be directly linked to a liposome containing the effector moiety of one of several types. The effector moiety(s) and/or the variant polypeptides or immunomodulatory proteins may also be bound to a solid surface.

In some embodiments, the variant polypeptides or immunomodulatory proteins of an IgSF conjugate and the effector moiety are both proteins and can be conjugated using techniques well known in the art. There are several hundred crosslinkers available that can conjugate two proteins. (See for example “Chemistry of Protein Conjugation and Crosslinking,” 1991, Shans Wong, CRC Press, Ann Arbor). The crosslinker is generally chosen based on the reactive functional groups available or inserted on the variant polypeptides or immunomodulatory proteins and/or effector moiety. In addition, if there are no reactive groups, a photoactivatible crosslinker can be used. In certain instances, it may be desirable to include a spacer between the variant polypeptides or immunomodulatory proteins and the effector moiety. Crosslinking agents known to the art include the homobifunctional agents: glutaraldehyde, dimethyladipimidate and Bi s(diazobenzidine) and the heterobifunctional agents: m Maleimidobenzoyl-N-Hydroxysuccinimide and Sulfo-m Maleimidobenzoyl-N-Hydroxysuccinimide.

In some embodiments, the variant polypeptides or immunomodulatory proteins of an IgSF conjugate may be engineered with specific residues for chemical attachment of the effector moiety. Specific residues used for chemical attachment of molecule known to the art include lysine and cysteine. The crosslinker is chosen based on the reactive functional groups inserted on the variant polypeptides or immunomodulatory proteins, and available on the effector moiety.

An IgSF conjugate may also be prepared using recombinant DNA techniques. In such a case a DNA sequence encoding the variant polypeptides or immunomodulatory proteins is fused to a DNA sequence encoding the effector moiety, resulting in a chimeric DNA molecule. The chimeric DNA sequence is transfected into a host cell that expresses the fusion protein. The fusion protein can be recovered from the cell culture and purified using techniques known in the art.

Examples of attaching an effector moiety, which is a label, to the variant polypeptides or immunomodulatory proteins include the methods described in Hunter, et al., Nature 144:945 (1962); David, et al., Biochemistry 13:1014 (1974); Pain, et al., J. Immunol. Meth. 40:219 (1981); Nygren, J. Histochem. and Cytochem. 30:407 (1982); Wensel and Meares, Radioimmunoimaging And Radioimmunotherapy, Elsevier, N.Y. (1983); and Colcher et al., “Use Of Monoclonal Antibodies As Radiopharmaceuticals For The Localization Of Human Carcinoma Xenografts In Athymic Mice”, Meth. Enzymol., 121:802-16 (1986).

The radio- or other labels may be incorporated in the conjugate in known ways. For example, the peptide may be biosynthesized or may be synthesized by chemical amino acid synthesis using suitable amino acid precursors involving, for example, fluorine-19 in place of hydrogen. Labels such as 99Tc or 123I, 186Re, 188Re and 111In can be attached via a cysteine residue in the peptide. Yttrium-90 can be attached via a lysine residue. The IODOGEN method (Fraker et al., Biochem. Biophys. Res. Commun. 80:49-57 (1978)) can be used to incorporate iodine-123. “Monoclonal Antibodies in Immunoscintigraphy” (Chatal, CRC Press 1989) describes other methods in detail.

Conjugates of the variant polypeptides or immunomodulatory proteins and a cytotoxic agent may be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14-labeled 1-p-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See, e.g., WO94/11026. The linker may be a “cleavable linker” facilitating release of the cytotoxic drug in the cell. For example, an acid-labile linker, peptidase-sensitive linker, photolabile linker, dimethyl linker or disulfide-containing linker (Chari et al., Cancer Research 52:127-131 (1992); U.S. Pat. No. 5,208,020) may be used.

The IgSF conjugates of the invention expressly contemplate, but are not limited to, drug conjugates prepared with cross-linker reagents: BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate) which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, Ill., U.S.A). See pages 467-498, 2003-2004 Applications Handbook and Catalog.

D. Transmembrane and Secretable Immunomodulatory Proteins and Engineered Cells

Provided herein are engineered cells which express the immunomodulatory variant PD-L2 polypeptides (alternatively, “engineered cells”). In some embodiments, the expressed immunomodulatory variant PD-L2 polypeptide is a transmembrane protein and is surface expressed. In some embodiments, the immunomodulatory variant PD-L2 polypeptide is expressed and secreted from the cell.

1. Transmembrane Immunomodulatory Proteins

In some embodiments, an immunomodulatory polypeptide comprising a variant PD-L2 can be a membrane bound protein. As described in more detail below, the immunomodulatory polypeptide can be a transmembrane immunomodulatory polypeptide comprising a variant PD-L2 in which is contained: an ectodomain containing at least one affinity modified IgSF domain (IgV or IgC), a transmembrane domain and, optionally, a cytoplasmic domain. In some embodiments, the transmembrane immunomodulatory protein can be expressed on the surface of an immune cell, such as a mammalian cell, including on the surface of a lymphocyte (e.g. T cell or NK cell) or antigen presenting cell. In some embodiments, the transmembrane immunomodulatory protein is expressed on the surface of a mammalian T-cell, including such T-cells as: a T helper cell, a cytotoxic T-cell (alternatively, cytotoxic T lymphocyte or CTL), a natural killer T-cell, a regulatory T-cell, a memory T-cell, or a gamma delta T-cell. In some embodiments, the mammalian cell is an antigen presenting cell (APC). Typically, but not exclusively, the ectodomain (alternatively, “extracellular domain”) of comprises the one or more amino acid variations (e.g. amino acid substitutions) of the variant PD-L2 of the invention. Thus, for example, in some embodiments a transmembrane protein will comprise an ectodomain that comprises one or more amino acid substitutions of a variant PD-L2 of the invention.

In some embodiments, the engineered cells express a variant PD-L2 polypeptides are transmembrane immunomodulatory polypeptides (TIPs) that can be a membrane protein such as a transmembrane protein. In typical embodiments, the ectodomain of a membrane protein comprises an extracellular domain or IgSF domain thereof of a variant PD-L2 provided herein in which is contained one or more amino acid substitutions in at least one IgSF domain as described. The transmembrane immunomodulatory proteins provided herein further contain a transmembrane domain linked to the ectodomain. In some embodiments, the transmembrane domain results in an encoded protein for cell surface expression on a cell. In some embodiments, the transmembrane domain is linked directly to the ectodomain. In some embodiments, the transmembrane domain is linked indirectly to the ectodomain via one or more linkers or spacers. In some embodiments, the transmembrane domain contains predominantly hydrophobic amino acid residues, such as leucine and valine.

In some embodiments, a full length transmembrane anchor domain can be used to ensure that the TIPs will be expressed on the surface of the engineered cell, such as engineered T cell. Conveniently, this could be from a particular native protein that is being affinity modified (e.g. PD-L2 or other native IgSF protein), and simply fused to the sequence of the first membrane proximal domain in a similar fashion as the native IgSF protein (e.g. PD-L2). In some embodiments, the transmembrane immunomodulatory protein comprises a transmembrane domain of the corresponding wild-type or unmodified IgSF member, such as a transmembrane domain contained in the sequence of amino acids set forth in SEQ ID NO:4 (Table 2). In some embodiments, the membrane bound form comprises a transmembrane domain of the corresponding wild-type or unmodified polypeptide, such as corresponding to residues 221-241 of SEQ ID NO:4.

In some embodiments, the transmembrane domain is a non-native transmembrane domain that is not the transmembrane domain of native PD-L2. In some embodiments, the transmembrane domain is derived from a transmembrane domain from another non-PD-L2 family member polypeptide that is a membrane-bound or is a transmembrane protein. In some embodiments, a transmembrane anchor domain from another protein on T cells can be used. In some embodiments, the transmembrane domain is derived from CD8. In some embodiments, the transmembrane domain can further contain an extracellular portion of CD8 that serves as a spacer domain. An exemplary CD8 derived transmembrane domain is set forth in SEQ ID NO: 266 or 1212 or a portion thereof containing the CD8 transmembrane domain. In some embodiments, the transmembrane domain is a synthetic transmembrane domain.

In some embodiments, the transmembrane immunomodulatory protein further contains an endodomain, such as a cytoplasmic signaling domain, linked to the transmembrane domain. In some embodiments, the cytoplasmic signaling domain induces cell signaling. In some embodiments, the endodomain of the transmembrane immunomodulatory protein comprises the cytoplasmic domain of the corresponding wild-type or unmodified polypeptide, such as a cytoplasmic domain contained in the sequence of amino acids set forth in SEQ ID NO:4 (see Table 2).

In some embodiments, a provided transmembrane immunomodulatory protein that is or comprises a variant PD-L2 comprises a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 216 and contains an ectodomain comprising at least one affinity-modified PD-L2 IgSF domain as described and a transmembrane domain. In some embodiments, the transmembrane immunomodulatory protein contains any one or more amino acid substitutions in an IgSF domain (e.g. IgV domain) as described, including any set forth in Table 1. In some embodiments, the transmembrane immunomodulatory protein can further comprise a cytoplasmic domain as described. In some embodiments, the transmembrane immunomodulatory protein can further contain a signal peptide. In some embodiments, the signal peptide is the native signal peptide of wild-type IgSF member, such as contained in the sequence of amino acids set forth in SEQ ID NO:4 (see e.g. Table 2).

Also provided is a nucleic acid molecule encoding such transmembrane immunomodulatory proteins. In some embodiments, a nucleic acid molecule encoding a transmembrane immunomodulatory protein comprises a nucleotide sequence that encodes a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NOS: 216 and contains an ectodomain comprising at least one affinity-modified IgSF domain as described, a transmembrane domain and, optionally, a cytoplasmic domain. In some embodiments, the nucleic acid molecule can further comprise a sequence of nucleotides encoding a signal peptide. In some embodiments, the signal peptide is the native signal peptide of the corresponding wild-type IgSF member (see e.g. Table 2).

In some embodiments, provided are CAR-related transmembrane immunomodulatory proteins in which the endodomain of a transmembrane immunomodulatory protein comprises a cytoplasmic signaling domain that comprises at least one ITAM (immunoreceptor tyrosine-based activation motif)-containing signaling domain. ITAM is a conserved motif found in a number of protein signaling domains involved in signal transduction of immune cells, including in the CD3-zeta chain (“CD3-z”) involved in T-cell receptor signal transduction. In some embodiments, the endodomain comprises at CD3-zeta signaling domain. In some embodiments, the CD3-zeta signaling domain comprises the sequence of amino acids set forth in SEQ ID NO: 243 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% to SEQ ID NO:243 and retains the activity of T cell signaling. In some embodiments, the endodomain of a CAR-related transmembrane immunomodulatory protein can further comprise a costimulatory signaling domain to further modulate immunomodulatory responses of the T-cell. In some embodiments, the costimulatory signaling domain is CD28, ICOS, 41BB or OX40. In some embodiments, the costimulatory signaling domain is a derived from CD28 or 4-1BB and comprises the sequence of amino acids set forth in any of SEQ ID NOS: 1213-1216 or a sequence of amino acids that exhibits at least 85%, 86%, 8′7%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% to SEQ ID NO:1213-1216 and retains the activity of T cell costimulatory signaling. In some embodiments, the provided CAR-related transmembrane immunomodulatory proteins have features of CARs to stimulate T cell signaling upon binding of an affinity modified IgSF domain to a cognate binding partner or counter structure. In some embodiments, upon specific binding by the affinity-modified IgSF domain to its counter structure can lead to changes in the immunological activity of the T-cell activity as reflected by changes in cytotoxicity, proliferation or cytokine production.

In some embodiments, the transmembrane immunomodulatory protein does not contain an endodomain capable of mediating cytoplasmic signaling. In some embodiments, the transmembrane immunomodulatory protein lacks the signal transduction mechanism of the wild-type or unmodified polypeptide and therefore does not itself induce cell signaling. In some embodiments, the transmembrane immunomodulatory protein lacks an intracellular (cytoplasmic) domain or a portion of the intracellular domain of the corresponding wild-type or unmodified polypeptide, such as a cytoplasmic signaling domain contained in the sequence of amino acids set forth in SEQ ID NO:4 (see Table 2). In some embodiments, the transmembrane immunomodulatory protein does not contain an ITIM (immunoreceptor tyrosine-based inhibition motif), such as contained in certain inhibitory receptors, including inhibitory receptors of the IgSF family (e.g. PD-1 or TIGIT). Thus, in some embodiments, the transmembrane immunomodulatory protein only contains the ectodomain and the transmembrane domain, such as any as described.

2. Secreted Immunomodulatory Proteins and Engineered Cells

In some embodiments, the PD-L2 variant immunomodulatory polypeptide containing any one or more of the amino acid mutations as described herein, is secretable, such as when expressed from a cell. Such a variant PD-L2 immunomodulatory protein does not comprise a transmembrane domain. In some embodiments, the variant PD-L2 immunomodulatory protein is not conjugated to a half-life extending moiety (such as an Fc domain or a multimerization domain). In some embodiments, the variant PD-L2 immunomodulatory protein comprises a signal peptide, e.g. an antibody signal peptide or other efficient signal sequence to get domains outside of cell. When the immunomodulatory protein comprises a signal peptide and is expressed by an engineered cell, the signal peptide causes the immunomodulatory protein to be secreted by the engineered cell. Generally, the signal peptide, or a portion of the signal peptide, is cleaved from the immunomodulatory protein with secretion. The immunomodulatory protein can be encoded by a nucleic acid (which can be part of an expression vector). In some embodiments, the immunomodulatory protein is expressed and secreted by a cell (such as an immune cell, for example a primary immune cell).

Thus, in some embodiments, there are provided variant PD-L2 immunomodulatory proteins that further comprises a signal peptide. In some embodiments, provided herein is a nucleic acid molecule encoding the variant PD-L2 immunomodulatory protein operably connected to a secretion sequence encoding the signal peptide.

A signal peptide is a sequence on the N-terminus of an immunomodulatory protein that signals secretion of the immunomodulatory protein from a cell. In some embodiments, the signal peptide is about 5 to about 40 amino acids in length (such as about 5 to about 7, about 7 to about 10, about 10 to about 15, about 15 to about 20, about 20 to about 25, or about 25 to about 30, about 30 to about 35, or about 35 to about 40 amino acids in length).

In some embodiments, the signal peptide is a native signal peptide from the corresponding wild-type PD-L2 (see Table 2). In some embodiments, the signal peptide is a non-native signal peptide. For example, in some embodiments, the non-native signal peptide is a mutant native signal peptide from the corresponding wild-type PD-L2, and can include one or more (such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) substitutions insertions or deletions. In some embodiments, the non-native signal peptide is a signal peptide or mutant thereof of a family member from the same IgSF family as the wild-type IgSF family member. In some embodiments, the non-native signal peptide is a signal peptide or mutant thereof from an IgSF family member from a different IgSF family that the wild-type IgSF family member. In some embodiments, the signal peptide is a signal peptide or mutant thereof from a non-IgSF protein family, such as a signal peptide from an immunoglobulin (such as IgG heavy chain or IgG-kappa light chain), a cytokine (such as interleukin-2 (IL-2), or CD33), a serum albumin protein (e.g. HSA or albumin), a human azurocidin preprotein signal sequence, a luciferase, a trypsinogen (e.g. chymotrypsinogen or trypsinogen) or other signal peptide able to efficiently secrete a protein from a cell. Exemplary signal peptides include any described in the Table 9.

TABLE 9 Exemplary Signal Peptides SEQ ID NO Signal Peptide Peptide Sequence SEQ ID NO: 245 HSA signal peptide MKWVTFISLLFLFSSAYS SEQ ID NO: 246 Ig kappa light chain MDMRAPAGIFGFLLVLFPGYRS SEQ ID NO: 247 human azurocidin preprotein signal sequence MTRLTVLALLAGLLASSRA SEQ ID NO: 248 IgG heavy chain signal peptide MELGLSWIFLLAILKGVQC SEQ ID NO: 249 IgG heavy chain signal peptide MELGLRWVFLVAILEGVQC SEQ ID NO: 250 IgG heavy chain signal peptide MKHLWFFLLLVAAPRWVLS SEQ ID NO: 251 IgG heavy chain signal peptide MDWTWRILFLVAAATGAHS SEQ ID NO: 252 IgG heavy chain signal peptide MDWTWRFLFVVAAATGVQS SEQ ID NO: 253 IgG heavy chain signal peptide MEFGLSWLFLVAILKGVQC SEQ ID NO: 254 IgG heavy chain signal peptide MEFGLSWVFLVALFRGVQC SEQ ID NO: 255 IgG heavy chain signal peptide MDLLHKNMKHLWFFLLLVAAPRWVLS SEQ ID NO: 256 IgG Kappa light chain signal sequences: MDMRVPAQLLGLLLLWLSGARC SEQ ID NO: 257 IgG Kappa light chain signal sequences: MKYLLPTAAAGLLLLAAQPAMA SEQ ID NO: 258 Gaussia luciferase MGVKVLFALICIAVAEA SEQ ED NO: 259 Human albumin MKWVTFISLLFLFSSAYS SEQ ED NO: 260 Human chymotlypsinogen MAFLWLLSCWALLGTTFG SEQ ED NO: 261 Human interleukin-2 MQLLSCIALILALV SEQ ED NO: 262 Human trypsinogen-2 MNLLLILTFVAAAVA

In some embodiments of a secretable variant PD-L2 immunomodulatory protein, the immunomodulatory protein comprises a signal peptide when expressed, and the signal peptide (or a portion thereof) is cleaved from the immunomodulatory protein upon secretion.

In some embodiments, the engineered cells express a variant PD-L2 polypeptides that are secreted from the cell. In some embodiments, such a variant PD-L2 polypeptide is encoded by a nucleic acid molecule encoding an immunomodulatory protein under the operable control of a signal sequence for secretion. In some embodiments, the encoded immunomodulatory protein is secreted when expressed from a cell. In some embodiments, the immunomodulatory protein encoded by the nucleic acid molecule does not comprise a transmembrane domain. In some embodiments, the immunomodulatory protein encoded by the nucleic acid molecule does not comprise a half-life extending moiety (such as an Fc domain or a multimerization domain). In some embodiments, the immunomodulatory protein encoded by the nucleic acid molecule comprises a signal peptide. In some embodiments, a nucleic acid of the invention further comprises nucleotide sequence that encodes a secretory or signal peptide operably linked to the nucleic acid encoding the immunomodulatory protein, thereby allowing for secretion of the immunomodulatory protein

3. Cells and Engineering Cells

Provided herein are engineered cells expressing any of the provided immunomodulatory polypeptides. In some embodiments, the engineered cells express on their surface any of the provided transmembrane immunomodulatory polypeptides. In some embodiments, the engineered cells express and are capable of or are able to secrete the immunomodulatory protein from the cells under conditions suitable for secretion of the protein. In some embodiments, the immunomodulatory protein is expressed on or in a lymphocyte such as a tumor infiltrating lymphocyte (TIL), T-cell or NK cell, or on a myeloid cell. In some embodiments, the engineered cells are antigen presenting cells (APCs). In some embodiments, the engineered cells are engineered mammalian T-cells or engineered mammalian antigen presenting cells (APCs). In some embodiments, the engineered T-cells or APCs are human or murine cells.

In some embodiments, engineered T-cells include, but are not limited to, T helper cell, cytotoxic T-cell (alternatively, cytotoxic T lymphocyte or CTL), natural killer T-cell, regulatory T-cell, memory T-cell, or gamma delta T-cell. In some embodiments, the engineered T cells are CD4+ or CD8+.

In some embodiments, the engineered APCs include, for example, MHC II expressing APCs such as macrophages, B cells, and dendritic cells, as well as artificial APCs (aAPCs) including both cellular and acellular (e.g., biodegradable polymeric microparticles) aAPCs. Artificial APCs (aAPCs) are synthetic versions of APCs that can act in a similar manner to APCs in that they present antigens to T-cells as well as activate them. Antigen presentation is performed by the MHC (Class I or Class II). In some embodiments, in engineered APCs such as aAPCs, the antigen that is loaded onto the MHC is, in some embodiments, a tumor specific antigen or a tumor associated antigen. The antigen loaded onto the MHC is recognized by a T-cell receptor (TCR) of a T cell, which, in some cases, can express PD-1 or other molecule recognized by the variant PD-L2 polypeptides provided herein. Materials which can be used to engineer an aAPC include: poly (glycolic acid), poly(lactic-co-glycolic acid), iron-oxide, liposomes, lipid bilayers, sepharose, and polystyrene.

In some embodiments a cellular aAPC can be engineered to contain a TIP and TCR agonist which is used in adoptive cellular therapy. In some embodiments, a cellular aAPC can be engineered to contain a TIP and TCR agonist which is used in ex vivo expansion of human T cells, such as prior to administration, e.g. for reintroduction into the patient. In some aspects, the aAPC may include expression of at least one anti-CD3 antibody clone, e.g. such as, for example, OKT3 and/or UCHT1. In some aspects, the aAPCs may be inactivated (e.g. irradiated). In some embodiment, the TIP can include any variant IgSF domain that exhibits binding affinity for a cognate binding partner on a T cell.

In some embodiments, an immunomodulatory protein provided herein, such as a transmembrane immunomodulatory protein or a secretable immunomodulatory protein, is co-expressed or engineered into a cell that expresses an antigen-binding receptor, such as a recombinant receptor, such as a chimeric antigen receptor (CAR) or T cell receptor (TCR). In some embodiments, the engineered cell, such as an engineered T cell, recognizes a desired antigen associated with cancer, inflammatory and autoimmune disorders, or a viral infection. In specific embodiments, the antigen-binding receptor contains an antigen-binding moiety that specifically binds a tumor specific antigen or a tumor associated antigen. In some embodiments, the engineered T-cell is a CAR (chimeric antigen receptor) T-cell that contains an antigen-binding domain (e.g. scFv) that specifically binds to an antigen, such as a tumor specific antigen or tumor associated antigen. In some embodiments, the TIP protein is expressed in an engineered T-cell receptor cell or an engineered chimeric antigen receptor cell. In such embodiments, the engineered cell co-expresses the TIP and the CAR or TCR. In some embodiments, the SIP protein is expressed in an engineered T-cell receptor cell or an engineered chimeric antigen receptor cell. In such embodiments, the engineered cell co-expresses the SIP and the CAR or TCR.

In some embodiments, the SIP protein is expressed in an engineered T-cell receptor cell or an engineered chimeric antigen receptor cell. In such embodiments, the engineered cell co-expresses the SIP and the CAR or TCR.

Chimeric antigen receptors (CARs) are recombinant receptors that include an antigen-binding domain (ectodomain), a transmembrane domain and an intracellular signaling region (endodomain) that is capable of inducing or mediating an activation signal to the T cell after the antigen is bound. In some example, CAR-expressing cells are engineered to express an extracellular single chain variable fragment (scFv) with specificity for a particular tumor antigen linked to an intracellular signaling part comprising an activating domain and, in some cases, a costimulatory domain. The costimulatory domain can be derived from, e.g., CD28, OX-40, 4-1BB/CD137, inducible T cell costimulator (ICOS), The activating domain can be derived from, e.g., CD3, such as CD3 zeta, epsilon, delta, gamma, or the like. In certain embodiments, the CAR is designed to have two, three, four, or more costimulatory domains. The CAR scFv can be designed to target an antigen exressed on a cell associated with a disease or condition, e.g. a tumor antigen, such as, for example, CD19, which is a transmembrane protein expressed by cells in the B cell lineage, including all normal B cells and B cell malignances, including but not limited to NHL, CLL, and non-T cell ALL. Example CAR+ T cell therapies and constructs are described in U.S. Patent Publication Nos. 2013/0287748, 2014/0227237, 2014/0099309, and 2014/0050708, and these references are incorporated by reference in their entirety.

In some aspects, the antigen-binding domain is an antibody or antigen-binding fragment thereof, such as a single chain fragment (scFv). In some embodiments, the antigen is expressed on a tumor or cancer cell. Exemplary of an antigen is CD19. Exemplary of a CAR is an anti-CD19 CAR, such as a CAR containing an anti-CD19 scFv set forth in SEQ ID NO:1163 or SEQ ID NO:1174. In some embodiments, the CAR further contains a spacer, a transmembrane domain, and an intracellular signaling domain or region comprising an ITAM signaling domain, such as a CD3zeta signaling domain. In some embodiments, the CAR further includes a costimulatory signaling domain. In some embodiments, the spacer and transmembrane domain are the hinge and transmembrane domain derived from CD8, such as having an exemplary sequence set forth in SEQ ID NO: 266, 1212, 2022 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:266, 1212, 2022. In some embodiments, the endodomain comprises at CD3-zeta signaling domain. In some embodiments, the CD3-zeta signaling domain comprises the sequence of amino acids set forth in SEQ ID NO: 243 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to SEQ ID NO:243 and retains the activity of T cell signaling. In some embodiments, the endodomain of a CAR can further comprise a costimulatory signaling domain or region to further modulate immunomodulatory responses of the T-cell. In some embodiments, the costimulatory signaling domain is or comprises a costimulatory region, or is derived from a costimulatory region, of CD28, ICOS, 41BB or OX40. In some embodiments, the costimulatory signaling domain is a derived from CD28 or 4-1BB and comprises the sequence of amino acids set forth in any of SEQ ID NOS: 1213-1216 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to SEQ ID NO: 1213-1216 and retains the activity of T cell costimulatory signaling.

In some embodiments, the construct encoding the CAR further encodes a second protein, such as a marker, e.g. detectable protein, separated from the CAR by a self-cleaving peptide sequence. In some embodiments, the self-cleaving peptide sequence is an F2A, T2A, E2A or P2A self-cleaving peptide. Exemplary sequences of a T2A self-cleaving peptide are set for the in any one of SEQ ID NOS: 1217, 1225 or 2029 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to any of SEQ ID NOS: 1217, 1225 or 2029. In some embodiments, the T2A is encoded by the sequence of nucleotides set forth in SEQ ID NO:1255 or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to any of SEQ ID NO: 1255. An exemplary sequence of a P2A self-cleaving peptide is set in SEQ ID NO: 2038 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to SEQ ID NOS: 2038. In some cases, a nucleic acid construct that encodes more than one P2A self-cleaving peptide (such as a P2A1 and P2A2), in which the nucleotide sequence P2A1 and P2A2 each encode the P2A set forth in SEQ ID NO:2038, the nucleotide sequence may be different to avoid recombination between sequences.

In some embodiments, the marker is a detectable protein, such as a fluorescent protein, e.g. a green fluorescent protein (GFP) or blue fluorescent protein (BFP). Exemplary sequences of a fluorescent protein marker are set forth in SEQ ID NO:1218, 2028, or 2035-2037 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to SEQ ID NO: 1218, 2028, or 2035-2037.

In some embodiments, the CAR has the sequence of amino acids set forth in any of SEQ ID NOS: 1208, 1219, 1220, 1221, 2023, 2024, 2026 or 2027 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to any one of SEQ ID NOS: 1208, 1219, 1220, 1221, 2023, 2024, 2026 or 2027. In some embodiments, the CAR is encoded by a sequence of nucleotides set forth in SEQ ID NO: 1223 or 2025 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to any one of SEQ ID NO: 1223 or 2025.

In another embodiment, the engineered T-cell possesses a TCR, including a recombinant or engineered TCR. In some embodiments, the TCR can be a native TCR. Those of skill in the art will recognize that generally native mammalian T-cell receptors comprise an alpha and a beta chain (or a gamma and a delta chain) involved in antigen specific recognition and binding. In some embodiments, the TCR is an engineered TCR that is modified. In some embodiments, the TCR of an engineered T-cell specifically binds to a tumor associated or tumor specific antigen presented by an APC.

In some embodiments, the immunomodulatory polypeptides, such as transmembrane immunomodulatory polypeptides or secretable immunomodulatory polypeptides, can be incorporated into engineered cells, such as engineered T cells or engineered APCs, by a variety of strategies such as those employed for recombinant host cells. A variety of methods to introduce a DNA construct into primary T cells are known in the art. In some embodiments, viral transduction or plasmid electroporation are employed. In typical embodiments, the nucleic acid molecule encoding the immunomodulatory protein, or the expression vector, comprises a signal peptide that localizes the expressed transmembrane immunomodulatory proteins to the cellular membrane or for secretion. In some embodiments, a nucleic acid encoding a transmembrane immunomodulatory proteins of the invention is sub-cloned into a viral vector, such as a retroviral vector, which allows expression in the host mammalian cell. The expression vector can be introduced into a mammalian host cell and, under host cell culture conditions, the immunomodulatory protein is expressed on the surface or is secreted.

In an exemplary example, primary T-cells can be purified ex vivo (CD4 cells or CD8 cells or both) and stimulated with an activation protocol consisting of various TCR/CD28 agonists, such as anti-CD3/anti-CD28 coated beads. After a 2 or 3 day activation process, a recombinant expression vector containing an immunomodulatory polypeptide can be stably introduced into the primary T cells through art standard lentiviral or retroviral transduction protocols or plasmid electroporation strategies. Cells can be monitored for immunomodulatory polypeptide expression by, for example, flow cytometry using anti-epitope tag or antibodies that cross-react with native parental molecule and polypeptides comprising variant PD-L2. T-cells that express the immunomodulatory polypeptide can be enriched through sorting with anti-epitope tag antibodies or enriched for high or low expression depending on the application.

Upon immunomodulatory polypeptide expression the engineered T-cell can be assayed for appropriate function by a variety of means. The engineered CAR or TCR co-expression can be validated to show that this part of the engineered T cell was not significantly impacted by the expression of the immunomodulatory protein. Once validated, standard in vitro cytotoxicity, proliferation, or cytokine assays (e.g., IFN-gamma expression) can be used to assess the function of engineered T-cells. Exemplary standard endpoints are percent lysis of the tumor line, proliferation of the engineered T-cell, or IFN-gamma protein expression in culture supernatants. An engineered construct which results in statistically significant increased lysis of tumor line, increased proliferation of the engineered T-cell, or increased IFN-gamma expression over the control construct can be selected for. Additionally, non-engineered, such as native primary or endogenous T-cells could also be incorporated into the same in vitro assay to measure the ability of the immunomodulatory polypeptide construct expressed on the engineered cells, such as engineered T-cells, to modulate activity, including, in some cases, to activate and generate effector function in bystander, native T-cells. Increased expression of activation markers such as CD69, CD44, or CD62L could be monitored on endogenous T cells, and increased proliferation and/or cytokine production could indicate desired activity of the immunomodulatory protein expressed on the engineered T cells.

In some embodiments, the similar assays can be used to compare the function of engineered T cells containing the CAR or TCR alone to those containing the CAR or TCR and a TIP construct. Typically, these in vitro assays are performed by plating various ratios of the engineered T cell and a “tumor” cell line containing the cognate CAR or TCR antigen together in culture. Standard endpoints are percent lysis of the tumor line, proliferation of the engineered T cell, or IFN-gamma production in culture supernatants. An engineered immunomodulatory protein which resulted in statistically significant increased lysis of tumor line, increased proliferation of the engineered T cell, or increased IFN-gamma production over the same TCR or CAR construct alone can be selected for. Engineered human T cells can be analyzed in immunocompromised mice, like the NSG strain, which lacks mouse T, NK and B cells. Engineered human T cells in which the CAR or TCR binds a target counter-structure on the xenograft and is co-expressed with the TIP affinity modified IgSF domain can be adoptively transferred in vivo at different cell numbers and ratios compared to the xenograft. For example, engraftment of CD19+ leukemia tumor lines containing a luciferase/GFP vector can be monitored through bioluminescence or ex vivo by flow cytometry. In a common embodiment, the xenograft is introduced into the murine model, followed by the engineered T cells several days later. Engineered T cells containing the immunomodulatory protein can be assayed for increased survival, tumor clearance, or expanded engineered T cells numbers relative to engineered T cells containing the CAR or TCR alone. As in the in vitro assay, endogenous, native (i.e., non-engineered) human T cells could be co-adoptively transferred to look for successful epitope spreading in that population, resulting in better survival or tumor clearance.

E. Infectious Agents Expressing Variant Polypeptides and Immunomodulatory Proteins

Also provided are infectious agents that contain nucleic acids encoding any of the variant polypeptides, such as PD-L2 vIgD polypeptides, including secretable or transmembrane immunomodulatory proteins described herein. In some embodiments, such infectious agents can deliver the nucleic acids encoding the variant immunomodulatory polypeptides described herein, such as PD-L2 vIgD polypeptides, to a target cell in a subject, e.g., immune cell and/or antigen-presenting cell (APC) or tumor cell in a subject. Also provided are nucleic acids contained in such infectious agents, and/or nucleic acids for generation or modification of such infectious agents, such as vectors and/or plasmids, and compositions containing such infectious agents.

In some embodiments, the infectious agent is a microorganism or a microbe. In some embodiments, the infectious agent is a virus or a bacterium. In some embodiments, the infectious agent is a virus. In some embodiments, the infectious agent is a bacterium. In some embodiments, such infectious agents can deliver nucleic acid sequences encoding any of the variant polypeptides, such as PD-L2 vIgD polypeptides, including secretable or transmembrane immunomodulatory proteins, described herein. Thus, in some embodiments, the cell in a subject that is infected or contacted by the infectious agents can be rendered to express on the cell surface or secrete, the variant immunomodulatory polypeptides. In some embodiments, the infectious agent can also deliver one or more other therapeutics or nucleic acids encoding other therapeutics to the cell and/or to an environment within the subject. In some embodiments, other therapeutics that can be delivered by the infectious agents include cytokines or other immunomodulatory molecules.

In some embodiments, the infectious agent, e.g., virus or bacteria, contains nucleic acid sequences that encode any of the variant polypeptides, such as PD-L2 vIgD polypeptides, including secretable or transmembrane immunomodulatory proteins, described herein, and by virtue of contact and/or infection of a cell in the subject, the cell expresses the variant polypeptides, such as PD-L2 vIgD polypeptides, including secretable or transmembrane immunomodulatory proteins, encoded by the nucleic acid sequences contained in the infectious agent. In some embodiments, the infectious agent can be administered to the subject. In some embodiments, the infectious agent can be contacted with cells from the subject ex vivo.

In some embodiments, the variant polypeptides, such as PD-L2 vIgD polypeptides, including transmembrane immunomodulatory proteins, expressed by the cell infected by the infectious agent is a transmembrane protein and is surface expressed. In some embodiments, the variant polypeptides, such as PD-L2 vIgD polypeptides, including secretable immunomodulatory proteins, expressed by the cell infected by the infectious agent is expressed and secreted from the cell. The transmembrane immunomodulatory protein or secreted immunomodulatory protein can be any described herein.

In some embodiments, the cells in the subject that are targeted by the infectious agent include a tumor cell, an immune cell, and/or an antigen-presenting cell (APC). In some embodiments, the infectious agent targets a cell in the tumor microenvironment (TME). In some embodiments, the infectious agent delivers the nucleic acids encoding the variant polypeptides, such as PD-L2 vIgD polypeptides, including secretable or transmembrane immunomodulatory proteins, to an appropriate cell (for example, an APC, such as a cell that displays a peptide/WIC complex on its cell surface, such as a dendritic cell) or tissue (e.g., lymphoid tissue) that will induce and/or augment the desired effect, e.g., immunomodulation and/or a specific cell-medicated immune response, e.g., CD4 and/or CD8 T cell response, which CD8 T cell response may include a cytotoxic T cell (CTL) response. In some embodiments, the infectious agent targets an APC, such as a dendritic cell (DC). In some embodiments, the nucleic acid molecule delivered by the infectious agents described herein include appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequences encoding the variant immunomodulatory polypeptides, in a particular target cell, e.g., regulatory elements such as promoters.

In some embodiments, the infectious agent that contains nucleic acid sequences encoding the immunomodulatory polypeptides can also contain nucleic acid sequences that encode one or more additional gene products, e.g., cytokines, prodrug converting enzymes, cytotoxins and/or detectable gene products. For example, in some embodiments, the infectious agent is an oncolytic virus and the virus can include nucleic acid sequences encoding additional therapeutic gene products (see, e.g., Kim et al., (2009) Nat Rev Cancer 9:64-71; Garcia-Aragoncillo et al., (2010) Curr Opin Mol Ther 12:403-411; see U.S. Pat. Nos. 7,588,767, 7,588,771, 7,662,398 and 7,754,221 and U.S. Pat. Publ. Nos. 2007/0202572, 2007/0212727, 2010/0062016, 2009/0098529, 2009/0053244, 2009/0155287, 2009/0117034, 2010/0233078, 2009/0162288, 2010/0196325, 2009/0136917 and 2011/0064650. In some embodiments, the additional gene product can be a therapeutic gene product that can result in death of the target cell (e.g., tumor cell) or gene products that can augment or boost or regulate an immune response (e.g., cytokine). Exemplary gene products also include among an anticancer agent, an anti-metastatic agent, an antiangiogenic agent, an immunomodulatory molecule, an immune checkpoint inhibitor, an antibody, a cytokine, a growth factor, an antigen, a cytotoxic gene product, a pro-apoptotic gene product, an anti-apoptotic gene product, a cell matrix degradative gene, genes for tissue regeneration and reprogramming human somatic cells to pluripotency, and other genes described herein or known to one of skill in the art. In some embodiments, the additional gene product is Granulocyte-macrophage colony-stimulating factor (GM-CSF).

1. Viruses

In some embodiments, the infectious agent is a virus. In some embodiments, the infectious agent is an oncolytic virus, or a virus that targets particular cells, e.g., immune cells. In some embodiments, the infectious agent targets a tumor cell and/or cancer cell in the subject. In some embodiments, the infectious agent targets an immune cell or an antigen-presenting cell (APC).

In some embodiments, the infectious agent is an oncolytic virus. Oncolytic viruses are viruses that accumulate in tumor cells and replicate in tumor cells. By virtue of replication in the cells, and optional delivery of nucleic acids encoding variant PD-L2 polypeptides or immunomodulatory polypeptides described herein, tumor cells are lysed, and the tumor shrinks and can be eliminated. Oncolytic viruses can also have a broad host and cell type range. For example, oncolytic viruses can accumulate in immunoprivileged cells or immunoprivileged tissues, including tumors and/or metastases, and also including wounded tissues and cells, thus allowing the delivery and expression of nucleic acids encoding the variant immunomodulatory polypeptides described herein in a broad range of cell types. Oncolytic viruses can also replicate in a tumor cell specific manner, resulting in tumor cell lysis and efficient tumor regression.

Exemplary oncolytic viruses include adenoviruses, adeno-associated viruses, herpes viruses, Herpes Simplex Virus, vesticular stomatic virus, Reovirus, Newcastle Disease virus, parvovirus, measles virus, vesticular stomatitis virus (VSV), Coxsackie virus and Vaccinia virus. In some embodiments, oncolytic viruses can specifically colonize solid tumors, while not infecting other organs, and can be used as an infectious agent to deliver the nucleic acids encoding the variant immunomodulatory polypeptides described herein to such solid tumors.

Oncolytic viruses for use in delivering the nucleic acids encoding variant PD-L2 polypeptides or immunomodulatory polypeptides described herein, can be any of those known to one of skill in the art and include, for example, vesicular stomatitis virus, see, e.g., U.S. Pat. Nos. 7,731,974, 7,153,510, 6,653,103 and U.S. Pat. Pub. Nos. 2010/0178684, 2010/0172877, 2010/0113567, 2007/0098743, 20050260601, 20050220818 and EP Pat. Nos. 1385466, 1606411 and 1520175; herpes simplex virus, see, e.g., U.S. Pat. Nos. 7,897,146, 7,731,952, 7,550,296, 7,537,924, 6,723,316, 6,428,968 and U.S. Pat. Pub. Nos., 2014/0154216, 2011/0177032, 2011/0158948, 2010/0092515, 2009/0274728, 2009/0285860, 2009/0215147, 2009/0010889, 2007/0110720, 2006/0039894, 2004/0009604, 2004/0063094, International Patent Pub. Nos., WO 2007/052029, WO 1999/038955; retroviruses, see, e.g., U.S. Pat. Nos. 6,689,871, 6,635,472, 5,851,529, 5,716,826, 5,716,613 and U.S. Pat. Pub. No. 20110212530; vaccinia viruses, see, e.g., 2016/0339066, and adeno-associated viruses, see, e.g., U.S. Pat. Nos. 8,007,780, 7,968,340, 7,943,374, 7,906,111, 7,927,585, 7,811,814, 7,662,627, 7,241,447, 7,238,526, 7,172,893, 7,033,826, 7,001,765, 6,897,045, and 6,632,670.

Oncolytic viruses also include viruses that have been genetically altered to attenuate their virulence, to improve their safety profile, enhance their tumor specificity, and they have also been equipped with additional genes, for example cytotoxins, cytokines, prodrug converting enzymes to improve the overall efficacy of the viruses (see, e.g., Kim et al., (2009) Nat Rev Cancer 9:64-71; Garcia-Aragoncillo et al., (2010) Curr Opin Mol Ther 12:403-411; see U.S. Pat. Nos. 7,588,767, 7,588,771, 7,662,398 and 7,754,221 and U.S. Pat. Publ. Nos. 2007/0202572, 2007/0212727, 2010/0062016, 2009/0098529, 2009/0053244, 2009/0155287, 2009/0117034, 2010/0233078, 2009/0162288, 2010/0196325, 2009/0136917 and 2011/0064650). In some embodiments, the oncolytic viruses can be those that have been modified so that they selectively replicate in cancerous cells, and, thus, are oncolytic. For example, the oncolytic virus is an adenovirus that has been engineered to have modified tropism for tumor therapy and also as gene therapy vectors. Exemplary of such is ONYX-015, H101 and Ad5ΔCR (Hallden and Portella (2012) Expert Opin Ther Targets, 16:945-58) and TNFerade (McLoughlin et al. (2005) Ann. Surg. Oncol., 12:825-30), or a conditionally replicative adenovirus Oncorine®.

In some embodiments, the infectious agent is a modified herpes simplex virus. In some embodiments, the infectious agent is a modified version of Talimogene laherparepvec (also known as T-Vec, Imlygic or OncoVex GM-CSF), that is modified to contain nucleic acids encoding any of the variant immunomodulatory polypeptides described herein, such as variant PD-L2 or immunomodulatory polypeptides described herein. In some embodiments, the infectious agent is a modified herpes simplex virus that is described, e.g., in WO 2007/052029, WO 1999/038955, US 2004/0063094, US 2014/0154216, or, variants thereof.

In some embodiments, the infectious agent is a virus that targets a particular type of cells in a subject that is administered the virus, e.g., a virus that targets immune cells or antigen-presenting cells (APCs). Dendritic cells (DCs) are essential APCs for the initiation and control of immune responses. DCs can capture and process antigens, migrate from the periphery to a lymphoid organ, and present the antigens to resting T cells in a major histocompatibility complex (MHC)-restricted fashion. In some embodiments, the infectious agent is a virus that specifically can target DCs to deliver nucleic acids encoding the variant PD-L2 polypeptides or immunomodulatory polypeptides for expression in DCs. In some embodiments, the virus is a lentivirus or a variant or derivative thereof, such as an integration-deficient lentiviral vector. In some embodiments, the virus is a lentivirus that is pseudotyped to efficiently bind to and productively infect cells expressing the cell surface marker dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN), such as DCs. In some embodiments, the virus is a lentivirus pseudotyped with a Sindbis virus E2 glycoprotein or modified form thereof, such as those described in WO 2013/149167. In some embodiments, the virus allows for delivery and expression of a sequence of interest (e.g., a nucleic acid encoding any of the variant PD-L2 polypeptides or immunomodulatory polypeptides described herein) to a DC. In some embodiments, the virus includes those described in WO 2008/011636 or US 2011/0064763, Tareen et al. (2014) Mol. Ther., 22:575-587, or variants thereof. Exemplary of a dendritic cell-tropic vector platform is ZVex™.

2. Bacteria

In some embodiments, the infectious agent is a bacterium. For example, in some embodiments, the bacteria can deliver nucleic acids encoding any of the variant PD-L2 polypeptides or immunomodulatory polypeptides described herein to a target cell in the subject, such as a tumor cell, an immune cell, an antigen-presenting cell and/or a phagocytic cell. In some embodiments, the bacterium can be preferentially targeted to a specific environment within a subject, such as a tumor microenvironment (TME), for expression and/or secretion of the variant immunomodulatory polypeptides and/or to target specific cells in the environment for expression of the variant immunomodulatory polypeptides.

In some embodiments, the bacterium delivers the nucleic acids to the cells via bacterial-mediated transfer of plasmid DNA to mammalian cells (also referred to as “bactofection”). For example, in some embodiments, delivery of genetic material is achieved through entry of the entire bacterium into target cells. In some embodiments, spontaneous or induced bacterial lysis can lead to the release of plasmid for subsequent eukaryotic cell expression. In some embodiments, the bacterium can deliver nucleic acids to non-phagocytic mammalian cells (e.g., tumor cells) and/or to phagocytic cells, e.g., certain immune cells and/or APCs. In some embodiments, the nucleic acids delivered by the bacterium can be transferred to the nucleus of the cell in the subject for expression. In some embodiments, the nucleic acids also include appropriate nucleic acid sequences necessary for the expression of the operably linked sequences encoding the variant immunomodulatory polypeptides in a particular host cell, e.g., regulatory elements such as promoters or enhancers. In some embodiments, the infectious agent that is a bacterium can deliver nucleic acids encoding the immunomodulatory proteins in the form of an RNA, such as a pre-made translation-competent RNA delivered to the cytoplasm of the target cell for translation by the target cell's machinery.

In some embodiments, the bacterium can replicate and lyse the target cells, e.g., tumor cells. In some embodiments, the bacterium can contain and/or release nucleic acid sequences and/or gene products in the cytoplasm of the target cells, thereby killing the target cell, e.g., tumor cell. In some embodiments, the infectious agent is bacterium that can replicate specifically in a particular environment in the subject, e.g., tumor microenvironment (TME). For example, in some embodiments, the bacterium can replicate specifically in anaerobic or hypoxic microenvironments. In some embodiments, conditions or factors present in particular environments, e.g., aspartate, serine, citrate, ribose or galactose produced by cells in the TME, can act as chemoattractants to attract the bacterium to the environment. In some embodiments, the bacterium can express and/or secrete the immunomodulatory proteins described herein in the environment, e.g., TME.

In some embodiments, the infectious agent is a bacterium that is a Listeria sp., a Bifidobacterium sp., an Escherichia sp., a Closteridium sp., a Salmonella sp., a Shigella sp., a Vibrio sp. or a Yersinia sp. In some embodiments, the bacterium is selected from among one or more of Listeria monocytogenes, Salmonella typhimurium, Salmonella choleraesuis, Escherichia coli, Vibrio cholera, Clostridium perfringens, Clostridium butyricum, Clostridium novyi, Clostridium acetobutylicum, Bifidobacterium infantis, Bifidobacterium longum and Bifidobacterium adolescentis. In some embodiments, the bacterium is an engineered bacterium. In some embodiments, the bacterium is an engineered bacterium such as those described in, e.g., Seow and Wood (2009) Molecular Therapy 17(5):767-777; Baban et al. (2010) Bioengineered Bugs 1:6, 385-394; Patyar et al. (2010) J Biomed Sci 17:21; Tangney et al. (2010) Bioengineered Bugs 1:4, 284-287; van Pijkeren et al. (2010) Hum Gene Ther. 21(4):405-416; WO 2012/149364; WO 2014/198002; U.S. Pat. Nos. 9,103,831; 9,453,227; US 2014/0186401; US 2004/0146488; US 2011/0293705; US 2015/0359909 and EP 3020816. The bacterium can be modified to deliver nucleic acid sequences encoding any of the variant immunomodulatory polypeptides, conjugates and/or fusions provided herein, and/or to express such variant immunomodulatory polypeptides in the subject.

F. Nucleic Acids, Vectors and Methods for Producing the Polypeptides or Cells

Provided herein are isolated or recombinant nucleic acids collectively referred to as “nucleic acids” which encode any of the various provided embodiments of the variant PD-L2 polypeptides or immunomodulatory polypeptides provided herein. In some embodiments, nucleic acids provided herein, including all described below, are useful in recombinant production (e.g., expression) of variant PD-L2 polypeptides or immunomodulatory polypeptides provided herein. In some embodiments, nucleic acids provided herein, including all described below, are useful in expression of variant PD-L2 polypeptides or immunomodulatory polypeptides provided herein in cells, such as in engineered cells, e.g. immune cells, or infectious agent cells. The nucleic acids provided herein can be in the form of RNA or in the form of DNA, and include mRNA, cRNA, recombinant or synthetic RNA and DNA, and cDNA. The nucleic acids provided herein are typically DNA molecules, and usually double-stranded DNA molecules. However, single-stranded DNA, single-stranded RNA, double-stranded RNA, and hybrid DNA/RNA nucleic acids or combinations thereof comprising any of the nucleotide sequences of the invention also are provided.

Also provided herein are recombinant expression vectors and recombinant host cells useful in producing the variant PD-L2 polypeptides or immunomodulatory polypeptides provided herein.

Also provided herein are engineered cells, such as engineered immune cells, containing any of the provided nucleic acid molecules or encoded variant PD-L2 polypeptides or immunomodulatory polypeptides, such as any of the transmembrane immunomodulatory polypeptides or secretable immunomodulatory polypeptides.

Also provided herein are infectious agents, such as bacterial or viral cells, containing any of the provided nucleic acid molecules or encoded variant PD-L2 polypeptides or immunomodulatory polypeptides, such as any of the transmembrane immunomodulatory polypeptides or secretable immunomodulatory polypeptides.

In any of the above provided embodiments, the nucleic acids encoding the immunomodulatory polypeptides provided herein can be introduced into cells using recombinant DNA and cloning techniques. To do so, a recombinant DNA molecule encoding a immunomodulatory polypeptide is prepared. Methods of preparing such DNA molecules are well known in the art. For instance, sequences coding for the peptides could be excised from DNA using suitable restriction enzymes. Alternatively, the DNA molecule could be synthesized using chemical synthesis techniques, such as the phosphoramidite method. Also, a combination of these techniques could be used. In some instances, a recombinant or synthetic nucleic acid may be generated through polymerase chain reaction (PCR). In some embodiments, a DNA insert can be generated encoding one or more variant PD-L2 polypeptides containing at least one affinity-modified IgSF domain and, in some embodiments, a signal peptide, a transmembrane domain and/or an endodomain in accord with the provided description. This DNA insert can be cloned into an appropriate transduction/transfection vector as is known to those of skill in the art. Also provided are expression vectors containing the nucleic acid molecules.

In some embodiments, the expression vectors are capable of expressing the immunomodulatory proteins in an appropriate cell under conditions suited to expression of the protein. In some aspects, nucleic acid molecule or an expression vector comprises the DNA molecule that encodes the immunomodulatory protein operatively linked to appropriate expression control sequences. Methods of effecting this operative linking, either before or after the DNA molecule is inserted into the vector, are well known. Expression control sequences include promoters, activators, enhancers, operators, ribosomal binding sites, start signals, stop signals, cap signals, polyadenylation signals, and other signals involved with the control of transcription or translation.

In some embodiments, expression of the immunomodulatory protein is controlled by a promoter or enhancer to control or regulate expression. The promoter is operably linked to the portion of the nucleic acid molecule encoding the variant polypeptide or immunomodulatory protein. In some embodiments, the promotor is a constitutively active promotor (such as a tissue-specific constitutively active promotor or other constitutive promotor). In some embodiments, the promotor is an inducible promotor, which may be responsive to an inducing agent (such as a T cell activation signal).

In some embodiments, a constitutive promoter is operatively linked to the nucleic acid molecule encoding the variant polypeptide or immunomodulatory protein. Exemplary constitutive promoters include the Simian vacuolating virus 40 (SV40) promoter, the cytomegalovirus (CMV) promoter, the ubiquitin C (UbC) promoter, and the EF-1 alpha (EF1a) promoter. In some embodiments, the constitutive promoter is tissue specific. For example, in some embodiments, the promoter allows for constitutive expression of the immunomodulatory protein in specific tissues, such as immune cells, lymphocytes, or T cells. Exemplary tissue-specific promoters are described in U.S. Pat. No. 5,998,205, including, for example, a fetoprotein, DF3, tyrosinase, CEA, surfactant protein, and ErbB2 promoters.

In some embodiments, an inducible promoter is operatively linked to the nucleic acid molecule encoding the variant polypeptide or immunomodulatory protein such that expression of the nucleic acid is controllable by controlling the presence or absence of the appropriate inducer of transcription. For example, the promoter can be a regulated promoter and transcription factor expression system, such as the published tetracycline-regulated systems or other regulatable systems (see, e.g. published International PCT Appl. No. WO 01/30843), to allow regulated expression of the encoded polypeptide. An exemplary regulatable promoter system is the Tet-On (and Tet-Off) system available, for example, from Clontech (Palo Alto, Calif.). This promoter system allows the regulated expression of the transgene controlled by tetracycline or tetracycline derivatives, such as doxycycline. Other regulatable promoter systems are known (see e.g., published U.S. Application No. 2002-0168714, entitled “Regulation of Gene Expression Using Single-Chain, Monomeric, Ligand Dependent Polypeptide Switches,” which describes gene switches that contain ligand binding domains and transcriptional regulating domains, such as those from hormone receptors).

In some embodiments, the promotor is responsive to an element responsive to T-cell activation signaling. Solely by way of example, in some embodiments, an engineered T cell comprises an expression vector encoding the immunomodulatory protein and a promotor operatively linked to control expression of the immunomodulatory protein. The engineered T cell can be activated, for example by signaling through an engineered T cell receptor (TCR) or a chimeric antigen rector (CAR), and thereby triggering expression and secretion of the immunomodulatory protein through the responsive promotor.

In some embodiments, an inducible promoter is operatively linked to the nucleic acid molecule encoding the immunomodulatory protein such that the immunomodulatory protein is expressed in response to a nuclear factor of activated T-cells (NFAT) or nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB). For example, in some embodiments, the inducible promoter comprises a binding site for NFAT or NF-κB. For example, in some embodiments, the promoter is an NFAT or NF-κB promoter or a functional variant thereof. Thus, in some embodiments, the nucleic acids make it possible to control the expression of immunomodulatory protein while also reducing or eliminating the toxicity of the immunomodulatory protein. In particular, engineered immune cells comprising the nucleic acids of the invention express and secrete the immunomodulatory protein only when the cell (e.g., a T-cell receptor (TCR) or a chimeric antigen receptor (CAR) expressed by the cell) is specifically stimulated by an antigen and/or the cell (e.g., the calcium signaling pathway of the cell) is non-specifically stimulated by, e.g., phorbol myristate acetate (PMA)/Ionomycin. Accordingly, the expression and, in some cases, secretion, of immunomodulatory protein can be controlled to occur only when and where it is needed (e.g., in the presence of an infectious disease-causing agent, cancer, or at a tumor site), which can decrease or avoid undesired immunomodulatory protein interactions.

In some embodiments, the nucleic acid encoding an immunomodulatory protein described herein comprises a suitable nucleotide sequence that encodes a NFAT promoter, NF-κB promoter, or a functional variant thereof. “NFAT promoter” as used herein means one or more NFAT responsive elements linked to a minimal promoter. “NF-κB promoter” refers to one or more NF-κB responsive elements linked to a minimal promoter. In some embodiments, the minimal promoter of a gene is a minimal human IL-2 promoter or a CMV promoter. The NFAT responsive elements may comprise, e.g., NFAT1, NFAT2, NFAT3, and/or NFAT4 responsive elements. The NFAT promoter, NF-κB promoter, or a functional variant thereof may comprise any number of binding motifs, e.g., at least two, at least three, at least four, at least five, or at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, or up to twelve binding motifs.

The resulting recombinant expression vector having the DNA molecule thereon is used to transform an appropriate host. This transformation can be performed using methods well known in the art. In some embodiments, a nucleic acid provided herein further comprises nucleotide sequence that encodes a secretory or signal peptide operably linked to the nucleic acid encoding an immunomodulatory polypeptide such that a resultant soluble immunomodulatory polypeptide is recovered from the culture medium, host cell, or host cell periplasm. In other embodiments, the appropriate expression control signals are chosen to allow for membrane expression of an immunomodulatory polypeptide. Furthermore, commercially available kits as well as contract manufacturing companies can also be utilized to make engineered cells or recombinant host cells provided herein.

In some embodiments, the resulting expression vector having the DNA molecule thereon is used to transform, such as transduce, an appropriate cell. The introduction can be performed using methods well known in the art. Exemplary methods include those for transfer of nucleic acids encoding the receptors, including via viral, e.g., retroviral or lentiviral, transduction, transposons, and electroporation. In some embodiments, the expression vector is a viral vector. In some embodiments, the nucleic acid is transferred into cells by lentiviral or retroviral transduction methods.

Any of a large number of publicly available and well-known mammalian host cells, including mammalian T-cells or APCs, can be used in the preparing the polypeptides or engineered cells. The selection of a cell is dependent upon a number of factors recognized by the art. These include, for example, compatibility with the chosen expression vector, toxicity of the peptides encoded by the DNA molecule, rate of transformation, ease of recovery of the peptides, expression characteristics, bio-safety and costs. A balance of these factors must be struck with the understanding that not all cells can be equally effective for the expression of a particular DNA sequence.

In some embodiments, the host cells can be a variety of eukaryotic cells, such as in yeast cells, or with mammalian cells such as Chinese hamster ovary (CHO) or HEK293 cells. In some embodiments, the host cell is a suspension cell and the polypeptide is engineered or produced in cultured suspension, such as in cultured suspension CHO cells, e.g. CHO-S cells. In some examples, the cell line is a CHO cell line that is deficient in DHFR (DHFR−), such as DG44 and DUXB11. In some embodiments, the cell is deficient in glutamine synthase (GS), e.g. CHO-S cells, CHOK1 SV cells, and CHOZN((R)) GS−/− cells. In some embodiments, the CHO cells, such as suspension CHO cells, may be CHO-S-2H2 cells, CHO-S-clone 14 cells, or ExpiCHO-S cells.

In some embodiments, host cells can also be prokaryotic cells, such as with E. coli. The transformed recombinant host is cultured under polypeptide expressing conditions, and then purified to obtain a soluble protein. Recombinant host cells can be cultured under conventional fermentation conditions so that the desired polypeptides are expressed. Such fermentation conditions are well known in the art. Finally, the polypeptides provided herein can be recovered and purified from recombinant cell cultures by any of a number of methods well known in the art, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, and affinity chromatography. Protein refolding steps can be used, as desired, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed in the final purification steps.

In some embodiments, the cell is an immune cells, such as any described above in connection with preparing engineered cells. In some embodiments, such engineered cells are primary cells. In some embodiments, the engineered cells are autologous to the subject. In some embodiment, the engineered cells are allogeneic to the subject. In some embodiments, the engineered cells are obtained from a subject, such as by leukapheresis, and transformed ex vivo for expression of the immunomodulatory polypeptide, e.g. transmembrane immunomodulatory polypeptide or secretable immunomodulatory polypeptide.

Also provided are nucleic acids encoding any of the variant immunomodulatory polypeptides contained in infectious agents described herein. In some embodiments, the infectious agents deliver the nucleic acids to a cell in the subject, and/or permit expression of the encoded variant polypeptides in the cell. Also provided are nucleic acids that are used to generate, produce or modify such infectious agents. For example, in some embodiments, provided are vectors and/or plasmids that contain nucleic acids encoding the variant immunomodulatory polypeptides, for generation of the infectious agents, delivery to the cells in a subject and/or expression of the variant immunomodulatory polypeptides in the cells in the subject.

In some embodiments, the provided nucleic acids are recombinant viral or bacterial vectors containing nucleic acid sequences encoding the variant immunomodulatory polypeptides. In some embodiments, the recombinant vectors can be used to produce an infectious agent that contains nucleic acid sequences encoding the variant immunomodulatory polypeptides and/or to be delivered to a target cell in the subject for expression by the target cell. In some embodiments, the recombinant vector is an expression vector. In some embodiments, the recombinant vector includes appropriate sequences necessary for generation and/or production of the infectious agent and expression in the target cell.

In some embodiments, the recombinant vector is a plasmid or cosmid. Plasmid or cosmid containing nucleic acid sequences encoding the variant immunomodulatory polypeptides, as described herein, is readily constructed using standard techniques well known in the art. For generation of the infectious agent, the vector or genome can be constructed in a plasmid form that can then be transfected into a packaging or producer cell line or a host bacterium. The recombinant vectors can be generated using any of the recombinant techniques known in the art. In some embodiments, the vectors can include a prokaryotic origin of replication and/or a gene whose expression confers a detectable or selectable marker such as a drug resistance for propagation and/or selection in prokaryotic systems.

In some embodiments, the recombinant vector is a viral vector. Exemplary recombinant viral vectors include a lentiviral vector genome, poxvirus vector genome, vaccinia virus vector genome, adenovirus vector genome, adenovirus-associated virus vector genome, herpes virus vector genome, and alpha virus vector genome. Viral vectors can be live, attenuated, replication conditional or replication deficient, non-pathogenic (defective), replication competent viral vector, and/or is modified to express a heterologous gene product, e.g., the variant immunomodulatory polypeptides provided herein. Vectors for generation of viruses also can be modified to alter attenuation of the virus, which includes any method of increasing or decreasing the transcriptional or translational load.

Exemplary viral vectors that can be used include modified vaccinia virus vectors (see, e.g., Guerra et al., J. Virol. 80:985-98 (2006); Tartaglia et al., AIDS Research and Human Retroviruses 8: 1445-47 (1992); Gheradi et al., J. Gen. Virol. 86:2925-36 (2005); Mayr et al., Infection 3:6-14 (1975); Hu et al., J. Virol. 75: 10300-308 (2001); U.S. Pat. Nos. 5,698,530, 6,998,252, 5,443,964, 7,247,615 and 7,368,116); adenovirus vector or adenovirus-associated virus vectors (see, e.g., Molin et al., J. Virol. 72:8358-61 (1998); Narumi et al., Am J. Respir. Cell Mol. Biol. 19:936-41 (1998); Mercier et al., Proc. Natl. Acad. Sci. USA 101:6188-93 (2004); U.S. Pat. Nos. 6,143,290; 6,596,535; 6,855,317; 6,936,257; 7,125,717; 7,378,087; 7,550,296); retroviral vectors including those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), ecotropic retroviruses, simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations (see, e.g., Buchscher et al., J. Virol. 66:2731-39 (1992); Johann et al., J. Virol. 66: 1635-40 (1992); Sommerfelt et al., Virology 176:58-59 (1990); Wilson et al., J. Virol. 63:2374-78 (1989); Miller et al., J. Virol. 65:2220-24 (1991); Miller et al., Mol. Cell Biol. 10:4239 (1990); Kolberg, NIH Res. 4:43 1992; Cornetta et al., Hum. Gene Ther. 2:215 (1991)); lentiviral vectors including those based upon Human Immunodeficiency Virus (HIV-1), HIV-2, feline immunodeficiency virus (FIV), equine infectious anemia virus, Simian Immunodeficiency Virus (SIV), and maedi/visna virus (see, e.g., Pfeifer et al., Annu. Rev. Genomics Hum. Genet. 2: 177-211 (2001); Zufferey et al., J. Virol. 72: 9873, 1998; Miyoshi et al., J. Virol. 72:8150, 1998; Philpott and Thrasher, Human Gene Therapy 18:483, 2007; Engelman et al., J. Virol. 69: 2729, 1995; Nightingale et al., Mol. Therapy, 13: 1121, 2006; Brown et al., J. Virol. 73:9011 (1999); WO 2009/076524; WO 2012/141984; WO 2016/011083; McWilliams et al., J. Virol. 77: 11150, 2003; Powell et al., J. Virol. 70:5288, 1996) or any, variants thereof, and/or vectors that can be used to generate any of the viruses described above. In some embodiments, the recombinant vector can include regulatory sequences, such as promoter or enhancer sequences, that can regulate the expression of the viral genome, such as in the case for RNA viruses, in the packaging cell line (see, e.g., U.S. Pat. Nos. 5,385,839 and 5,168,062).

In some embodiments, the recombinant vector is an expression vector, e.g., an expression vector that permits expression of the encoded gene product when delivered into the target cell, e.g., a cell in the subject, e.g., a tumor cell, an immune cell and/or an APC. In some embodiments, the recombinant expression vectors contained in the infectious agent are capable of expressing the immunomodulatory proteins in the target cell in the subject, under conditions suited to expression of the protein.

In some aspects, nucleic acids or an expression vector comprises a nucleic acid sequence that encodes the immunomodulatory protein operatively linked to appropriate expression control sequences. Methods of affecting this operative linking, either before or after the nucleic acid sequence encoding the immunomodulatory protein is inserted into the vector, are well known. Expression control sequences include promoters, activators, enhancers, operators, ribosomal binding sites, start signals, stop signals, cap signals, polyadenylation signals, and other signals involved with the control of transcription or translation. The promoter can be operably linked to the portion of the nucleic acid sequence encoding the immunomodulatory protein. In some embodiments, the promotor is a constitutively active promotor in the target cell (such as a tissue-specific constitutively active promotor or other constitutive promotor). For example, the recombinant expression vector may also include, lymphoid tissue-specific transcriptional regulatory elements (TRE) such as a B lymphocyte, T lymphocyte, or dendritic cell specific TRE. Lymphoid tissue specific TRE are known in the art (see, e.g., Thompson et al., Mol. Cell. Biol. 12:1043-53 (1992); Todd et al., J. Exp. Med. 177:1663-74 (1993); Penix et al., J. Exp. Med. 178:1483-96 (1993)). In some embodiments, the promotor is an inducible promotor, which may be responsive to an inducing agent (such as a T cell activation signal). In some embodiments, nucleic acids delivered to the target cell in the subject, e.g., tumor cell, immune cell and/or APC, can be operably linked to any of the regulatory elements described above.

In some embodiments, the vector is a bacterial vector, e.g., a bacterial plasmid or cosmid. In some embodiments, the bacterial vector is delivered to the target cell, e.g., tumor cells, immune cells and/or APCs, via bacterial-mediated transfer of plasmid DNA to mammalian cells (also referred to as “bactofection”). In some embodiments, the delivered bacterial vector also contains appropriate expression control sequences for expression in the target cells, such as a promoter sequence and/or enhancer sequences, or any regulatory or control sequences described above. In some embodiments, the bacterial vector contains appropriate expression control sequences for expression and/or secretion of the encoded variant polypeptides in the infectious agent, e.g., the bacterium.

In some embodiments, polypeptides provided herein can also be made by synthetic methods. Solid phase synthesis is the preferred technique of making individual peptides since it is the most cost-effective method of making small peptides. For example, well known solid phase synthesis techniques include the use of protecting groups, linkers, and solid phase supports, as well as specific protection and deprotection reaction conditions, linker cleavage conditions, use of scavengers, and other aspects of solid phase peptide synthesis. Peptides can then be assembled into the polypeptides as provided herein.

IV. METHODS OF ASSESSING ACTIVITY IMMUNE MODULATION OF VARIANT PD-L2 POLYPEPTIDES AND IMMUNOMODULATORY PROTEINS

In some embodiments, the variant PD-L2 polypeptides provided herein (e.g. full-length and/or specific binding fragments or conjugates, stack constructs or fusion thereof, engineered cells or infectious agents) exhibit immunomodulatory activity to modulate T cell activation. In some embodiments, PD-L2 polypeptides modulate IFN-gamma expression in a T cell assay relative to a wild-type or unmodified PD-L2 control. In some cases, modulation of IFN-gamma expression can increase or decrease IFN-gamma expression relative to the control. Assays to determine specific binding and IFN-gamma expression are well-known in the art and include the MLR (mixed lymphocyte reaction) assays measuring interferon-gamma cytokine levels in culture supernatants (Wang et al., Cancer Immunol Res. 2014 September: 2(9):846-56), SEB (staphylococcal enterotoxin B) T cell stimulation assay (Wang et al., Cancer Immunol Res. 2014 September: 2(9):846-56), and anti-CD3 T cell stimulation assays (Li and Kurlander, J Transl Med. 2010: 8: 104).

In some embodiments, a variant PD-L2 polypeptide can in some embodiments increase or, in alternative embodiments, decrease IFN-gamma (interferon-gamma) expression in a primary T-cell assay relative to a wild-type PD-L2 control. In some embodiments, such activity may depend on whether the variant PD-L2 polypeptide is provided in a form for antagonist activity or in a form for agonist activity. In some embodiments, a variant PD-L2 polypeptide or immunomodulatory protein is an antagonist of the inhibitory receptor, such as blocks an inhibitory signal in the cell that may occur to decrease response to activating stimuli, e.g. CD3 and/or CD28 costimulatory signal or a mitogenic signal. Those of skill will recognize that different formats of the primary T-cell assay used to determine an increase or decrease in IFN-gamma expression exist.

In assaying for the ability of a variant PD-L2 to increase or decrease IFN-gamma expression in a primary T-cell assay, a Mixed Lymphocyte Reaction (MLR) assay can be used. In some embodiments, a variant PD-L2 polypeptide or immunomodulatory protein provided in antagonist form, such as soluble form, e.g. variant PD-L2-Fc or secretable immunomodulatory protein, block activity of the PD-1 inhibitory receptor and thereby increase MLR activity in the assay, such as observed by increased production of IFN-gamma in the assay. In some embodiments, a variant PD-L2 polypeptide or immunomodulatory protein provided in agonist form, such as a localizing vIgD stack or conjugate containing a tumor-localizing moiety or an engineered cell expressing a transmembrane immunomodulatory protein as provided, may stimulate activity of the PD-1 inhibitory receptor and thereby decrease MLR activity, such as evidenced by decreased IFN-gamma production. In some embodiments, a variant PD-L2 polypeptide or immunomodulatory protein provided in agonist form, such as a localizing vIgD stack or conjugate containing a tumor-localizing moiety or an engineered cell expressing a transmembrane immunomodulatory protein as provided, may block activity of the PD-1 inhibitory receptor and thereby increase MLR activity, such as increase IFN-gamma production.

Alternatively, in assaying for the ability of a variant PD-L2 to modulate an increase or decrease IFN-gamma expression in a primary T-cell assay, a co-immobilization assay can be used. In a co-immobilization assay, a TCR signal, provided in some embodiments by anti-CD3 antibody, is used in conjunction with a co-immobilized variant PD-L2 to determine the ability to increase or decrease IFN-gamma expression relative to a PD-L2 unmodified or wild-type control. In some embodiments, a variant PD-L2 polypeptide or immunomodulatory protein, e.g. a co-immobilized variant PD-L2-Fc, decreases IFN-gamma production in a co-immobilization assay.

In some embodiments, in assaying for the ability of a variant PD-L2 to modulate an increase or decrease IFN-gamma expression a T cell reporter assay can be used. In some embodiments, the T cell is a Jurkat T cell line or is derived from Jurkat T cell lines. In reporter assays, the reporter cell line (e.g. Jurkat reporter cell) also is generated to overexpress an inhibitory receptor that is the cognate binding partner of the variant IgSF domain polypeptide. For example, in the case of a variant PD-L2, the reporter cell line (e.g. Jurkat reporter cell) is generated to overexpress PD-1. In some embodiments, the reporter T cells also contain a reporter construct containing an inducible promoter responsive to T cell activation operably linked to a reporter. In some embodiments, the reporter is a fluorescent or luminescent reporter. In some embodiments, the reporter is luciferase. In some embodiments, the promoter is responsive to CD3 signaling. In some embodiments, the promoter is an NFAT promoter. In some embodiments, the promoter is responsive to costimulatory signaling, e.g. CD28 costimulatory signaling. In some embodiments, the promoter is an IL-2 promoter.

In aspects of a reporter assay, a reporter cell line is stimulated, such as by co-incubation with antigen presenting cells (APCs) expressing the wild-type ligand of the inhibitory receptor, e.g. PD-L2. In some embodiments, the APCs are artificial APCs. Artificial APCs are well known to a skilled artisan. In some embodiments, artificial APCs are derived from one or more mammalian cell line, such as K562, CHO or 293 cells.

In some embodiments, the Jurkat reporter cells are co-incubated with artificial APCs overexpressing the inhibitory ligand in the presence of the variant IgSF domain molecule or immunomodulatory protein, e.g., variant PD-L2 polypeptide or immunomodulatory protein. In some embodiments, reporter expression is monitored, such as by determining the luminescence or fluorescence of the cells. In some embodiments, normal interactions between its inhibitory receptor and ligand result in a repression of or decrease in the reporter signal, such as compared to control, e.g. reporter expression by co-incubation of control T cells and APCs in which the inhibitory receptor and ligand interaction is not present, e.g. APCs that do not overexpress PD-L2. In some embodiments, a variant PD-L2 polypeptide or immunomodulatory protein provided herein antagonizes the interaction, e.g. when provided in soluble form as a variant PD-L2-Fc or when expressed from the APC as a secretable immunomodulatory protein, thereby resulting in an increase in the reporter signal compared to the absence of the variant PD-L2 polypeptide or immunomodulatory protein. In some cases, certain formats of a variant PD-L2 polypeptide or immunomodulatory protein as provided herein may provide an agonist activity, thereby decreasing reporter expression compared to the absence of the variant PD-L2 polypeptide or immunomodulatory protein.

Use of proper controls is known to those of skill in the art, however, in the aforementioned embodiments, a control typically involves use of the unmodified PD-L2, such as a wild-type of native PD-L2 isoform from the same mammalian species from which the variant PD-L2 was derived or developed. In some embodiments, the wild-type or native PD-L2 is of the same form or corresponding form as the variant. For example, if the variant PD-L2 is a soluble form containing a variant ECD fused to an Fc protein, then the control is a soluble form containing the wild-type or native ECD of PD-L2 fused to the Fc protein. Irrespective of whether the binding affinity and/or selectivity to PD-1 is increased or decreased, a variant PD-L2 in some embodiments will increase IFN-gamma expression and, in alternative embodiments, decrease IFN-gamma expression in a T-cell assay relative to a wild-type PD-L2 control.

In some embodiments, a variant PD-L2 polypeptide or immunomodulatory protein, increases IFN-gamma expression (i.e., protein expression) relative to a wild-type or unmodified PD-L2 control by at least: 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or higher. In other embodiments, a variant PD-L2 or immunomodulatory protein decreases IFN-gamma expression (i.e. protein expression) relative to a wild-type or unmodified PD-L2 control by at least: 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or higher. In some embodiments, the wild-type PD-L2 control is murine PD-L2, such as would typically be used for a variant PD-L2 altered in sequence from that of a wild-type murine PD-L2 sequence. In some embodiments, the wild-type PD-L2 control is human PD-L2, such as would typically be used for a variant PD-L2 altered in sequence from that of a wild-type human PD-L2 sequence.

V. PHARMACEUTICAL FORMULATIONS

Provided herein are compositions containing any of the variant PD-L2 polypeptides, immunomodulatory proteins, conjugates, engineered cells or infectious agents described herein. The pharmaceutical composition can further comprise a pharmaceutically acceptable excipient. For example, the pharmaceutical composition can contain one or more excipients for modifying, maintaining or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption, or penetration of the composition. In some aspects, a skilled artisan understands that a pharmaceutical composition containing cells may differ from a pharmaceutical composition containing a protein.

In some embodiments, the pharmaceutical composition is a solid, such as a powder, capsule, or tablet. For example, the components of the pharmaceutical composition can be lyophilized. In some embodiments, the solid pharmaceutical composition is reconstituted or dissolved in a liquid prior to administration.

In some embodiments, the pharmaceutical composition is a liquid, for example variant PD-L2 polypeptides dissolved in an aqueous solution (such as physiological saline or Ringer's solution). In some embodiments, the pH of the pharmaceutical composition is between about 4.0 and about 8.5 (such as between about 4.0 and about 5.0, between about 4.5 and about 5.5, between about 5.0 and about 6.0, between about 5.5 and about 6.5, between about 6.0 and about 7.0, between about 6.5 and about 7.5, between about 7.0 and about 8.0, or between about 7.5 and about 8.5).

In some embodiments, the pharmaceutical composition comprises a pharmaceutically-acceptable excipient, for example a filler, binder, coating, preservative, lubricant, flavoring agent, sweetening agent, coloring agent, a solvent, a buffering agent, a chelating agent, or stabilizer. Examples of pharmaceutically-acceptable fillers include cellulose, dibasic calcium phosphate, calcium carbonate, microcrystalline cellulose, sucrose, lactose, glucose, mannitol, sorbitol, maltol, pregelatinized starch, corn starch, or potato starch. Examples of pharmaceutically-acceptable binders include polyvinylpyrrolidone, starch, lactose, xylitol, sorbitol, maltitol, gelatin, sucrose, polyethylene glycol, methyl cellulose, or cellulose. Examples of pharmaceutically-acceptable coatings include hydroxypropyl methylcellulose (HPMC), shellac, corn protein zein, or gelatin. Examples of pharmaceutically-acceptable disintegrants include polyvinylpyrrolidone, carboxymethyl cellulose, or sodium starch glycolate. Examples of pharmaceutically-acceptable lubricants include polyethylene glycol, magnesium stearate, or stearic acid. Examples of pharmaceutically-acceptable preservatives include methyl parabens, ethyl parabens, propyl paraben, benzoic acid, or sorbic acid. Examples of pharmaceutically-acceptable sweetening agents include sucrose, saccharine, aspartame, or sorbitol. Examples of pharmaceutically-acceptable buffering agents include carbonates, citrates, gluconates, acetates, phosphates, or tartrates.

In some embodiments, the pharmaceutical composition further comprises an agent for the controlled or sustained release of the product, such as injectable microspheres, bio-erodible particles, polymeric compounds (polylactic acid, polyglycolic acid), beads, or liposomes.

In some embodiments, the pharmaceutical composition is sterile. Sterilization may be accomplished by filtration through sterile filtration membranes or radiation. Where the composition is lyophilized, sterilization using this method may be conducted either prior to or following lyophilization and reconstitution. The composition for parenteral administration may be stored in lyophilized form or in solution. In addition, parenteral compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

In some embodiments, provided are pharmaceutical compositions containing the transmembrane immunomodulatory proteins, including engineered cells expressing such transmembrane immunomodulatory proteins. In some embodiments, the pharmaceutical compositions and formulations include one or more optional pharmaceutically acceptable carrier or excipient. Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. Compositions of the present invention are preferably formulated for intravenous administration.

Such a formulation may, for example, be in a form suitable for intravenous infusion. A pharmaceutically acceptable carrier may be a pharmaceutically acceptable material, composition, or vehicle that is involved in carrying or transporting cells of interest from one tissue, organ, or portion of the body to another tissue, organ, or portion of the body. For example, the carrier may be a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or some combination thereof. Each component of the carrier must be “pharmaceutically acceptable” in that it must be compatible with the other ingredients of the formulation. It also must be suitable for contact with any tissue, organ, or portion of the body that it may encounter, meaning that it must not carry a risk of toxicity, irritation, allergic response, immunogenicity, or any other complication that excessively outweighs its therapeutic benefits.

VI. ARTICLES OF MANUFACTURE AND KITS

Also provided herein are articles of manufacture comprising the pharmaceutical compositions described herein in suitable packaging. Suitable packaging for compositions (such as ophthalmic compositions) described herein are known in the art, and include, for example, vials (such as sealed vials), vessels, ampules, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. These articles of manufacture may further be sterilized and/or sealed.

Further provided are kits comprising the pharmaceutical compositions (or articles of manufacture) described herein, which may further comprise instruction(s) on methods of using the composition, such as uses described herein. The kits described herein may also include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for performing any methods described herein.

VII. THERAPEUTIC APPLICATIONS

Provided herein are methods using the provided pharmaceutical compositions containing a variant PD-L2 polypeptide, immunomodulatory protein, conjugate, engineered cell or infectious agent described herein, for modulating an immune response, including in connection with treating a disease or condition in a subject, such as in a human patient. The pharmaceutical compositions described herein (including pharmaceutical composition comprising the variant PD-L2 polypeptides, the immunomodulatory proteins, the conjugates, the engineered cells or the infectious agents described herein) can be used in a variety of therapeutic applications, such as the treatment of a disease. For example, in some embodiments the pharmaceutical composition is used to treat inflammatory or autoimmune disorders, cancer, organ transplantation, viral infections, and/or bacterial infections in a mammal. The pharmaceutical composition can modulate (e.g. increase or decrease) an immune response to treat the disease.

In some embodiments, the provided methods are applicable to therapeutic administration of variant PD-L2 polypeptides, the immunomodulatory proteins, the conjugates, the engineered cells and infectious agents described herein. It is within the level of a skilled artisan, in view of the provided disclosure, to choose a format for the indication depending on the type of modulation of the immune response, e.g. increase or decrease that is desired.

In some embodiments, a pharmaceutical composition provided herein that stimulates the immune response is administered, which can be useful, for example, in the treatment of cancer, viral infections, or bacterial infections. In some embodiments, the pharmaceutical composition contains a variant PD-L2 polypeptide in a format that exhibits antagonist activity of its cognate binding partner PD-1 and/or that inhibits costimulatory signaling via PD-1. Exemplary formats of PD-L2 polypeptide for use in connection with such therapeutic applications include, for example, a variant PD-L2 polypeptide that is soluble (e.g. variant PD-L2-Fc fusion protein), an immunomodulatory protein or “stack” of a variant PD-L2 polypeptide and another IgSF domain, including soluble forms thereof that are Fc fusions, an engineered cell expressing a secretable immunomodulatory protein, or an infectious agent comprising a nucleic acid molecule encoding a secretable immunomodulatory protein, such as for expression and secretion of the secretable immunomodulatory protein in an infected cell (e.g. tumor cell or APC, e.g. dendritic cell). Among such methods are methods carried out by delivery of a variant PD-L2 polypeptide in a soluble format. Exemplary soluble formats are described herein, including formats in which an extracellular portion of a variant PD-L2 polypeptide containing an affinity modified IgSF domain (e.g. IgV) is linked, directly or indirectly, to a multimerization domain, e.g. an Fc domain or region. In some embodiments, such a therapeutic agent is a variant PD-L2-Fc fusion protein.

The provided methods to modulate an immune response can be used to treat a disease or condition, such as a tumor or cancer. In some embodiments, the pharmaceutical composition can be used to inhibit growth of mammalian cancer cells (such as human cancer cells). A method of treating cancer can include administering an effective amount of any of the pharmaceutical compositions described herein to a subject with cancer. The effective amount of the pharmaceutical composition can be administered to inhibit, halt, or reverse progression of cancers, including cancers that are sensitive to modulation of immunological activity, such as by the provided variants or immunomodulatory proteins. Human cancer cells can be treated in vivo, or ex vivo. In ex vivo treatment of a human patient, tissue or fluids containing cancer cells are treated outside the body and then the tissue or fluids are reintroduced back into the patient. In some embodiments, the cancer is treated in a human patient in vivo by administration of the therapeutic composition into the patient. Thus, the present invention provides ex vivo and in vivo methods to inhibit, halt, or reverse progression of the tumor, or otherwise result in a statistically significant increase in progression-free survival (i.e., the length of time during and after treatment in which a patient is living with cancer that does not get worse), or overall survival (also called “survival rate;” i.e., the percentage of people in a study or treatment group who are alive for a certain period of time after they were diagnosed with or treated for cancer) relative to treatment with a control.

The cancers that can be treated by the pharmaceutical compositions and the treatment methods described herein include, but are not limited to, melanoma, bladder cancer, hematological malignancies (leukemia, lymphoma, myeloma), liver cancer, brain cancer, renal cancer, breast cancer, pancreatic cancer (adenocarcinoma), colorectal cancer, lung cancer (small cell lung cancer and non-small-cell lung cancer), spleen cancer, cancer of the thymus or blood cells (i.e., leukemia), prostate cancer, testicular cancer, ovarian cancer, uterine cancer, gastric carcinoma, a musculoskeletal cancer, a head and neck cancer, a gastrointestinal cancer, a germ cell cancer, or an endocrine and neuroendocrine cancer. In some embodiments, the cancer is Ewing's sarcoma. In some embodiments, the cancer is a lymphoma, lymphoid leukemia, myeloid leukemia, cervical cancer, neuroblastoma, or multiple myeloma.

In some embodiments, the pharmaceutical composition is administered as a monotherapy (i.e., as a single agent) or as a combination therapy (i.e., in combination with one or more additional anticancer agents, such as a chemotherapeutic drug, a cancer vaccine, or an immune checkpoint inhibitor. In some embodiments, the pharmaceutical composition can also be administered with radiation therapy. In some aspects of the present disclosure, the immune checkpoint inhibitor is nivolumab, tremelimumab, pembrolizumab, ipilimumab, or the like.

In some embodiments, the pharmaceutical composition suppresses an immune response, which can be useful in the treatment of inflammatory or autoimmune disorders, or organ transplantation. In some embodiments, the pharmaceutical composition contains a variant PD-L2 polypeptide in a format that exhibits agonist activity of its cognate binding partner PD-1 and/or that stimulates inhibitory signaling via PD-1. Exemplary formats of a PD-L2 polypeptide for use in connection with such therapeutic applications include, for example, an immunomodulatory protein or “stack” of a variant PD-L2 polypeptide and an IgSF domain or variant thereof that localizes to a cell or tissue of an inflammatory environment, a conjugate containing a variant PD-L2 polypeptide linked to a moiety that localizes to a cell or tissue of an inflammatory environment, an engineered cell expressing a transmembrane immunomodulatory protein, or an infectious agent comprising a nucleic acid molecule encoding a transmembrane immunomodulatory protein, such as for expression of the transmembrane immunomodulatory protein in an infected cell.

The provided methods to modulate an immune response can be used to treat a disease or condition, such as an inflammatory or autoimmune disorder. In some embodiments, the inflammatory or autoimmune disorder is Antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis, a vasculitis, an autoimmune skin disease, transplantation, a Rheumatic disease, an inflammatory gastrointestinal disease, an inflammatory eye disease, an inflammatory neurological disease, an inflammatory pulmonary disease, an inflammatory endocrine disease, or an autoimmune hematological disease.

In some embodiments, the inflammatory and autoimmune disorders that can be treated by the pharmaceutical composition described herein is Addison's Disease, allergies, alopecia areata, Alzheimer's, antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis, ankylosing spondylitis, antiphospholipid syndrome (Hughes Syndrome), asthma, atherosclerosis, rheumatoid arthritis, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome, autoimmune myocarditis, autoimmune oophoritis, autoimmune orchitis, azoospermia, Behcet's Disease, Berger's Disease, bullous pemphigoid, cardiomyopathy, cardiovascular disease, celiac Sprue/coeliac disease, chronic fatigue immune dysfunction syndrome (CFIDS), chronic idiopathic polyneuritis, chronic inflammatory demyelinating, polyradicalneuropathy (CIDP), chronic relapsing polyneuropathy (Guillain-Barré syndrome), Churg-Strauss Syndrome (CSS), cicatricial pemphigoid, cold agglutinin disease (CAD), COPD (chronic obstructive pulmonary disease), CREST syndrome, Crohn's disease, dermatitis, herpetiformus, dermatomyositis, diabetes, discoid lupus, eczema, epidermolysis bullosa acquisita, essential mixed cryoglobulinemia, Evan's Syndrome, exopthalmos, fibromyalgia, Goodpasture's Syndrome, Graves' Disease, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), IgA nephropathy, immunoproliferative disease or disorder, inflammatory bowel disease (IBD), interstitial lung disease, juvenile arthritis, juvenile idiopathic arthritis (JIA), Kawasaki's Disease, Lambert-Eaton Myasthenic Syndrome, lichen planus, lupus nephritis, lymphocytic hypophysitis, Meniere's Disease, Miller Fish Syndrome/acute disseminated encephalomyeloradiculopathy, mixed connective tissue disease, multiple sclerosis (MS), muscular rheumatism, myalgic encephalomyelitis (ME), myasthenia gravis, ocular inflammation, pemphigus foliaceus, pemphigus vulgaris, pernicious anaemia, polyarteritis nodosa, polychondritis, polyglandular syndromes (Whitaker's syndrome), polymyalgia rheumatica, polymyositis, primary agammaglobulinemia, primary biliary cirrhosis/autoimmune cholangiopathy, psoriasis, psoriatic arthritis, Raynaud's Phenomenon, Reiter's Syndrome/reactive arthritis, restenosis, rheumatic fever, rheumatic disease, sarcoidosis, Schmidt's syndrome, scleroderma, Sjörgen's Syndrome, stiff-man syndrome, systemic lupus erythematosus (SLE), systemic scleroderma, Takayasu arteritis, temporal arteritis/giant cell arteritis, thyroiditis, Type 1 diabetes, ulcerative colitis, uveitis, vasculitis, vitiligo, interstitial bowel disease or Wegener's Granulomatosis. In some embodiments, the inflammatory or autoimmune disorder is selected from interstitial bowel disease, transplant, Crohn's disease, ulcerative colitis, multiple sclerosis, asthma, rheumatoid arthritis, and psoriasis.

In some embodiments, the pharmaceutical composition is administered to modulate an autoimmune condition. For example, suppressing an immune response can be beneficial in methods for inhibiting rejection of a tissue, cell, or organ transplant from a donor by a recipient. Accordingly, in some embodiments, the pharmaceutical compositions described herein are used to limit or prevent graft-related or transplant related diseases or disorders, such as graft versus host disease (GVHD). In some embodiments, the pharmaceutical compositions are used to suppress autoimmune rejection of transplanted or grafted bone marrow, organs, skin, muscle, neurons, islets, or parenchymal cells.

Pharmaceutical compositions comprising engineered cells and the methods described herein can be used in adoptive cell transfer applications. In some embodiments, cell compositions comprising engineered cells can be used in associated methods to, for example, modulate immunological activity in an immunotherapy approach to the treatment of, for example, a mammalian cancer or, in other embodiments the treatment of autoimmune disorders. The methods employed generally comprise a method of contacting a TIP of the present invention with a mammalian cell under conditions that are permissive to specific binding of the affinity modified IgSF domain and modulation of the immunological activity of the mammalian cell. In some embodiments, immune cells (such as tumor infiltrating lymphocytes (TILs), T-cells (including CD8+ or CD4+ T-cells), or APCs) are engineered to express as a membrane protein and/or as a soluble variant PD-L2 polypeptide, immunomodulatory protein, or conjugate as described herein. The engineered cells can then be contact a mammalian cell, such as an APC, a second lymphocyte or tumor cell in which modulation of immunological activity is desired and under conditions that are permissive of specific binding of the affinity modified IgSF domain to a counter-structure on the mammalian cell such that immunological activity can be modulated in the mammalian cell. Cells can be contacted in vivo or ex vivo.

In some embodiments, the engineered cells are autologous cells. In other embodiments, the cells are allogeneic. In some embodiments, the cells are autologous engineered cells reinfused into the mammal from which it was isolated. In some embodiments, the cells are allogenic engineered cells infused into the mammal. In some embodiments, the cells are harvested from a patient's blood or tumor, engineered to express a polypeptide (such as the variant PD-L2 polypeptide, immunomodulatory protein, or conjugate as described herein), expanded in an in vitro culture system (for example, by stimulating the cells), and reinfused into the patient to mediate tumor destruction. In some embodiments, the methods are conducted by adoptive cell transfer wherein cells expressing the TIP (e.g., a T-cell) are infused back into the patient. In some embodiments, the therapeutic compositions and methods of the invention are used in the treatment of a mammalian patient of cancers such as lymphoma, lymphoid leukemia, myeloid leukemia, cervical cancer, neuroblastoma, or multiple myeloma.

In some embodiments, the pharmaceutical composition is administered to a subject. Generally, dosages and routes of administration of the pharmaceutical composition are determined according to the size and condition of the subject, according to standard pharmaceutical practice. For example, the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models such as mice, rats, rabbits, dogs, pigs, or monkeys. An animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans. The exact dosage will be determined in light of factors related to the subject requiring treatment. Dosage and administration are adjusted to provide sufficient levels of the active compound or to maintain the desired effect. Factors that may be taken into account include the severity of the disease state, the general health of the subject, the age, weight, and gender of the subject, time and frequency of administration, drug combination(s), reaction sensitivities, and response to therapy.

Long-acting pharmaceutical compositions may be administered every 3 to 4 days, every week, or biweekly depending on the half-life and clearance rate of the particular formulation. The frequency of dosing will depend upon the pharmacokinetic parameters of the molecule in the formulation used. Typically, a composition is administered until a dosage is reached that achieves the desired effect. The composition may therefore be administered as a single dose, or as multiple doses (at the same or different concentrations/dosages) over time, or as a continuous infusion. Further refinement of the appropriate dosage is routinely made. Appropriate dosages may be ascertained through use of appropriate dose-response data. A number of biomarkers or physiological markers for therapeutic effect can be monitored including T cell activation or proliferation, cytokine synthesis or production (e.g., production of TNF-α, IFN-γ, IL-2), induction of various activation markers (e.g., CD25, IL-2 receptor), inflammation, joint swelling or tenderness, serum level of C-reactive protein, anti-collagen antibody production, and/or T cell-dependent antibody response(s).

In some embodiments, the pharmaceutical composition is administered to a subject through any route, including orally, transdermally, by inhalation, intravenously, intra-arterially, intramuscularly, direct application to a wound site, application to a surgical site, intraperitoneally, by suppository, subcutaneously, intradermally, transcutaneously, by nebulization, intrapleurally, intraventricularly, intra-articularly, intraocularly, or intraspinally.

In some embodiments, the dosage of the pharmaceutical composition is a single dose or a repeated dose. In some embodiments, the doses are given to a subject once per day, twice per day, three times per day, or four or more times per day. In some embodiments, about 1 or more (such as about 2 or more, about 3 or more, about 4 or more, about 5 or more, about 6 or more, or about 7 or more) doses are given in a week. In some embodiments, multiple doses are given over the course of days, weeks, months, or years. In some embodiments, a course of treatment is about 1 or more doses (such as about 2 or more does, about 3 or more doses, about 4 or more doses, about 5 or more doses, about 7 or more doses, about 10 or more doses, about 15 or more doses, about 25 or more doses, about 40 or more doses, about 50 or more doses, or about 100 or more doses).

In some embodiments, an administered dose of the pharmaceutical composition is about 1 μg of protein per kg subject body mass or more (such as about 2 μg of protein per kg subject body mass or more, about 5 μg of protein per kg subject body mass or more, about 10 of protein per kg subject body mass or more, about 25 μg of protein per kg subject body mass or more, about 50 μg of protein per kg subject body mass or more, about 100 μg of protein per kg subject body mass or more, about 250 μg of protein per kg subject body mass or more, about 500 of protein per kg subject body mass or more, about 1 mg of protein per kg subject body mass or more, about 2 mg of protein per kg subject body mass or more, or about 5 mg of protein per kg subject body mass or more).

In some embodiments, a therapeutic amount of a cell composition is administered. Typically, precise amount of the compositions of the present invention to be administered can be determined by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject). It can generally be stated that a pharmaceutical composition comprising engineered cells, e.g. T cells, as described herein may be administered at a dosage of 10⁴ to 10⁹ cells/kg body weight, such as 10⁵ to 10⁶ cells/kg body weight, including all integer values within those ranges. Engineered cell compositions, such as T cell compositions, may also be administered multiple times at these dosages. The cells can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al, New Eng. J. of Med. 319: 1676, 1988). The optimal dosage and treatment regime for a particular patient can readily be determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly.

In some embodiments, the pharmaceutical composition contains infectious agents containing nucleic acid sequences encoding the immunomodulatory variant polypeptides. In some embodiments, the pharmaceutical composition contains a dose of infectious agents suitable for administration to a subject that is suitable for treatment. In some embodiments, the pharmaceutical composition contains an infectious agent that is a virus, at a single or multiple dosage amount, of between about between or between about 1×10⁵ and about 1×10¹² plaque-forming units (pfu), 1×10⁶ and 1×10¹⁰ pfu, or 1×10⁷ and 1×10¹⁰ pfu, each inclusive, such as at least or at least about or at about 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹ pfu or about 1×10¹⁰ pfu. In some embodiments, the pharmaceutical composition can contain a virus concentration of from or from about 10⁵ to about 10¹⁰ pfu/mL, for example, 5×10⁶ to 5×10⁹ or 1×10⁷ to 1×10⁹ pfu/mL, such as at least or at least about or at about 10⁶ pfu/mL, 10⁷ pfu/mL, 10⁸ pfu/mL or 10⁹ pfu/mL. In some embodiments, the pharmaceutical composition contains an infectious agent that is a bacterium, at a single or multiple dosage amount, of between about between or between about 1×10³ and about 1×10⁹ colony-forming units (cfu), 1×10⁴ and 1×10⁹ cfu, or 1×10⁵ and 1×10⁷ cfu, each inclusive, such as at least or at least about or at about 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸ or 1×10⁹ cfu. In some embodiments, the pharmaceutical composition can contain a bacterial concentration of from or from about 10³ to about 10⁸ cfu/mL, for example, 5×10⁵ to 5×10⁷ or 1×10⁶ to 1×10⁷ cfu/mL, such as at least or at least about or at about 10⁵ cfu/mL, 10⁶ cfu/mL, 10⁷ cfu/mL or 10⁸ cfu/mL.

A variety of means are known for determining if administration of a therapeutic composition of the invention sufficiently modulates immunological activity by eliminating, sequestering, or inactivating immune cells mediating or capable of mediating an undesired immune response; inducing, generating, or turning on immune cells that mediate or are capable of mediating a protective immune response; changing the physical or functional properties of immune cells; or a combination of these effects. Examples of measurements of the modulation of immunological activity include, but are not limited to, examination of the presence or absence of immune cell populations (using flow cytometry, immunohistochemistry, histology, electron microscopy, polymerase chain reaction (PCR)); measurement of the functional capacity of immune cells including ability or resistance to proliferate or divide in response to a signal (such as using T-cell proliferation assays and pepscan analysis based on 3H-thymidine incorporation following stimulation with anti-CD3 antibody, anti-T-cell receptor antibody, anti-CD28 antibody, calcium ionophores, PMA (phorbol 12-myristate 13-acetate) antigen presenting cells loaded with a peptide or protein antigen; B cell proliferation assays); measurement of the ability to kill or lyse other cells (such as cytotoxic T cell assays); measurements of the cytokines, chemokines, cell surface molecules, antibodies and other products of the cells (e.g., by flow cytometry, enzyme-linked immunosorbent assays, Western blot analysis, protein microarray analysis, immunoprecipitation analysis); measurement of biochemical markers of activation of immune cells or signaling pathways within immune cells (e.g., Western blot and immunoprecipitation analysis of tyrosine, serine or threonine phosphorylation, polypeptide cleavage, and formation or dissociation of protein complexes; protein array analysis; DNA transcriptional, profiling using DNA arrays or subtractive hybridization); measurements of cell death by apoptosis, necrosis, or other mechanisms (e.g., annexin V staining, TUNEL assays, gel electrophoresis to measure DNA laddering, histology; fluorogenic caspase assays, Western blot analysis of caspase substrates); measurement of the genes, proteins, and other molecules produced by immune cells (e.g., Northern blot analysis, polymerase chain reaction, DNA microarrays, protein microarrays, 2-dimensional gel electrophoresis, Western blot analysis, enzyme linked immunosorbent assays, flow cytometry); and measurement of clinical symptoms or outcomes such as improvement of autoimmune, neurodegenerative, and other diseases involving self-proteins or self-polypeptides (clinical scores, requirements for use of additional therapies, functional status, imaging studies) for example, by measuring relapse rate or disease severity (using clinical scores known to the ordinarily skilled artisan) in the case of multiple sclerosis, measuring blood glucose in the case of type I diabetes, or joint inflammation in the case of rheumatoid arthritis.

VIII. EXEMPLARY EMBODIMENTS

Among the provided embodiments are:

1. A variant PD-L2 polypeptide, comprising an IgV domain or a specific binding fragment thereof, an IgC domain or a specific binding fragment thereof, or both, wherein the variant PD-L2 polypeptide comprises one or more amino acid modifications at one or more positions in an unmodified PD-L2 or a specific binding fragment thereof corresponding to position(s) selected from 2, 12, 13, 15, 18, 23, 24, 28, 31, 32, 33, 36, 37, 39, 44, 45, 46, 47, 48, 58, 59, 65, 67, 69, 71, 72, 73, 74, 75, 76, 77, 82, 85, 86, 89, or 91 with reference to numbering of SEQ ID NO:31.

2. The variant PD-L2 polypeptide of embodiment 1, wherein the amino acid modification is an amino acid substitution, insertion or deletion.

3. The variant PD-L2 polypeptide of embodiment 1 or embodiment 2, wherein the unmodified PD-L2 is a mammalian PD-L2 or a specific binding fragment thereof.

4. The variant PD-L2 polypeptide of any of embodiments 1-3, wherein the unmodified PD-L2 is a human PD-L2 or a specific binding fragment thereof.

5. The variant PD-L2 polypeptide of any one of embodiments 1-4, wherein the unmodified PD-L2 comprises (i) the sequence of amino acids set forth in SEQ ID NO:31, (ii) a sequence of amino acids that has at least 95% sequence identity to SEQ ID NO:31; or (iii) a portion thereof comprising an IgV domain or IgC domain or specific binding fragments thereof or both.

6. The variant PD-L2 polypeptide of any one of embodiments 1-5, wherein:

the specific binding fragment of the IgV domain or IgC domain has a length of at least 50, 60, 70, 80, 90, 100, 110 or more amino acids; or

the specific binding fragment of the IgV domain comprises a length that is at least 80% of the length of the IgV domain set forth as amino acids 24-130 of SEQ ID NO:4 and/or the specific binding fragment of the IgC domain comprises a length that is at least 80% of the length of the IgC domain set forth as amino acids 122-203 of SEQ ID NO:4.

7. The variant PD-L2 polypeptide of any one of embodiments 1-6, wherein:

the variant PD-L2 comprises up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid modifications, optionally amino acid substitutions, insertions and/or deletions; or

the variant PD-L2 polypeptide comprises a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:31 or a specific binding fragment thereof.

8. The variant PD-L2 polypeptide of any of embodiments 1-7, wherein the variant PD-L2 polypeptide exhibits altered binding to the ectodomain of PD-1 or RGMb compared to the unmodified PD-L2.

9. The variant PD-L2 polypeptide of any of embodiments 1-8, wherein the variant PD-L2 polypeptide exhibits altered binding to the ectodomain of PD-1 compared to the unmodified PD-L2.

10. The variant PD-L2 polypeptide of embodiment 8 or embodiment 9, wherein the altered binding is altered binding affinity and/or altered binding selectivity.

11. The variant PD-L2 polypeptide of any of embodiments 1-10, wherein the one or more amino acid modifications are selected from F2L, 112V, I13V, H15Q, N18D, N24S, C23S, G28V, N24D, V31A, V31M, N32D, L33P, L33H, L33F, I36V, T37A, S48C, S39I, E44D, N45S, D46E, T47A, E58G, E59G, K65R, S67L, H69L, P71S, Q72H, V73A, Q74R, R76G, D77N, Q82R, I85F, I86T, V89D, W91R, or a conservative amino acid substitution thereof.

12. The variant PD-L2 polypeptide of any of embodiments 1-11, wherein the one or more amino acid modifications are selected from among H15Q, N24D, E44D, V89D, Q82R/V89D,

E59G/Q82R, S39I/V89D, S67L/V89D, S67L/I85F, S67L/I86T, H15Q/K65R, H15Q/Q72H/V89D, H15Q/S67L/R76G, H15Q/R76G/I85F, H15Q/T47A/Q82R, H15Q/Q82R/V89D, H15Q/C23S/I86T, H15Q/S39I/I86T, E44D/V89D/W91R, I13V/S67L/V89D, H15Q/S67L/I86T, I13V/H15Q/S67L/I86T, I13V/H15Q/E44D/V89D, I13V/S39I/E44D/Q82R/V89D, I13V/E44D/Q82R/V89D, I13V/Q72H/R76G/I86T, I13V/H15Q/R76G/I85F, H15Q/S39I/R76G/V89D, H15Q/S67L/R76G/I85F, H15Q/T47A/Q72H/R76G/I86T, H15Q/T47A/Q72H/R76G, I13V/H15Q/T47A/Q72H/R76G, H15Q/E44D/R76G/I85F, H15Q/S39I/S67L/V89D, H15Q/N32D/S67L/V89D, N32D/S67L/V89D, H15Q/S67L/Q72H/R76G/V89D, H15Q/Q72H/Q74R/R76G/I86T, G28V/Q72H/R76G/I86T, I13V/H15Q/S39I/E44D/S67L, E44D/S67L/Q72H/Q82R/V89D, H15Q/V89D, H15Q/T47A, I13V/H15Q/Q82R, I13V/H15Q/V89D, I13V/S67L/Q82R/V89D, I13V/H15Q/Q82R/V89D, H15Q/V31M/S67L/Q82R/V89D, I13V/H15Q/T47A/Q82R, I13V/H15Q/V31A/N45S/Q82R/V89D, H15Q/T47A/H69L/Q82R/V89D, I13V/H15Q/T47A/H69L/R76G/V89D, I12V/I13V/H15Q/T47A/Q82R/V89D, I13V/H15Q/R76G/D77N/Q82R/V89D, I13V/H15Q/T47A/R76G/V89D, I13V/H15Q/T47A/Q82R/V89D, I13V/H15Q/N24D/Q82R/V89D, I13V/H15Q/I36V/T47A/S67L/V89D, H15Q/T47A/K65R/S67L/Q82R/V89D, H15Q/L33P/T47A/S67L/P71S/V89D, I13V/H15Q/Q72H/R76G/I86T, H15Q/T47A/S67L/Q82R/V89D, F2L/H15Q/D46E/T47A/Q72H/R76G/Q82R/V89D, I13V/H15Q/L33F/T47A/Q82R/V89D, I13V/H15Q/T47A/E58G/S67L/Q82R/V89D, H15Q/N24S/T47A/Q72H/R76G/V89D, I13V/H15Q/E44V/T47A/Q82R/V89D, H15Q/N18D/T47A/Q72H/V73A/R76G/I86T/V89D, I13V/H15Q/T37A/E44D/S48C/S67L/Q82R/V89D, H15Q/L33H/S67L/R76G/Q82R/V89D, I13V/H15Q/T47A/Q72H/R76G/I86T, H15Q/S39I/E44D/Q72H/V75G/R76G/Q82R/V89D, H15Q/T47A/S67L/R76G/Q82R/V89D, or I13V/H15Q/T47A/S67L/Q72H/R76G/Q82R/V89D.

13. The variant PD-L2 polypeptide of any of embodiments 1-12, wherein the one or more amino acid substitutions correspond to position(s) selected from 13, 15, 39, 44, 47 67, 72, 76, 82, 85, 86, or 89, optionally wherein the one or more amino acid substitutions are selected from I13V, H15Q, E44D, T47A, S67L, Q72H, R76G, Q82R, I85F, I86T, V89D, or a conservative amino acid substitution thereof.

14. The variant PD-L2 polypeptide of any of embodiments 1-13, wherein the one or more amino acid substitutions correspond to position(s) selected from 13, 15, 44, 47, 67, 72, 76, 82, 86, or 89, optionally wherein the one or more amino acid substitutions are selected from I13V, H15Q, E44D, T47A, S67L, Q72H, R76G, Q82R, I86T, V89D, or a conservative amino acid substitution thereof.

15. The variant PD-L2 polypeptide of any of embodiments 1-14, wherein the variant PD-L2 polypeptide comprises amino acid modifications I13V/H15Q, I13V/T47A, I13V/S67L, I13V/Q72H, I13V/Q72H, I13V/R76G, I13V/Q82R, I13V/I86T, I13V/V89D, H15Q/T47A, H15Q/S67L, H15Q/Q72H, H15Q/Q72H, H15Q/R76G, H15Q/Q82R, H15Q/I86T, H15Q/V89D, T47A/S67L, T47A/Q72H, T47A/Q72H, T47A/R76G, T47A/Q82R, T47A/I86T, T47A/V89D, S67L/Q72H, S67L/Q72H, S67L/R76G, S67L/Q82R, S67L/I86T, S67L/V89D, Q72H/R76G, Q72H/Q82R, Q72H/I86T, Q72H/V89D, R76G/Q82R, R76G/I86T, R76G/V89D, Q82R/I86T, Q82R/V89D or I86T/V89D.

16. The variant PD-L2 polypeptide of any of embodiments 1-11, wherein the variant PD-L2 polypeptide comprises amino acid modifications H15Q/S62L/Q82R, H15Q/S62L/V89D, H15Q/Q82R/V89D, or S62L/Q82R/V89D.

17. The variant PD-L2 polypeptide of any of embodiments 1-16, wherein the variant PD-L2 polypeptide comprises amino acid modifications H15Q/T47A/K65R/S67L/Q82R/V89D.

18. The variant PD-L2 polypeptide of any of embodiments 1-17, wherein the variant PD-L2 polypeptide comprises or consists of the IgV domain or a specific fragment thereof and the IgC domain or a specific fragment thereof.

19. The variant PD-L2 polypeptide of any of embodiments 1-18, comprising or consisting of the sequence of amino acids set forth in any of SEQ ID NOS: 56-106, 108-114, 116-132 or a specific binding fragment thereof, or a sequence of amino acids that exhibits at least 95% sequence identity to any of SEQ ID NOS: 56-106, 108-114, 116-132 or a specific binding fragment thereof and that contains the one or more of the amino acid substitutions thereof.

20. The variant PD-L2 polypeptide of any of embodiments 1-17, wherein the variant PD-L2 polypeptide comprises or consists of the IgV domain or a specific binding fragment thereof.

21. The variant PD-L2 polypeptide of any of embodiments 1-17 and 20, wherein the IgV domain or specific binding fragment thereof is the only PD-L2 portion of the variant PD-L2 polypeptide.

22. The variant PD-L2 polypeptide of any of embodiments 1-21, comprising or consisting of the sequence of amino acids set forth in any of SEQ ID NOS: 133-183, 185-191, 193-209, 268-318, 320-343 or a specific binding fragment thereof, or a sequence of amino acids that exhibits at least 95% sequence identity to any of SEQ ID NOS: 133-183, 185-191, 193-209, 268-318, 320-343 or a specific binding fragment thereof and that comprises the one or more of the amino acid modifications thereof.

23. The variant PD-L2 polypeptide of any of embodiments 1-17, wherein the IgC domain or specific binding fragment thereof is the only PD-L2 portion of the variant PD-L2 polypeptide.

24. The variant PD-L2 polypeptide of any of embodiments 1-23, wherein the variant PD-L2 polypeptide specifically binds to the ectodomain of PD-1 or RGMb with increased affinity compared to the binding of the unmodified PD-L2 to the ectodomain of PD-1 or RGMb.

25. The variant PD-L2 polypeptide of any of embodiments 1-24, wherein the variant PD-L2 polypeptide specifically binds to the ectodomain of PD-1 with increased affinity compared to the binding of the unmodified PD-L2 to the ectodomain of PD-1.

26. The variant PD-L2 polypeptide of any of embodiments 1-25, wherein the variant PD-L2 polypeptide specifically binds to the ectodomain of PD-1 and the ectodomain of RGMb each with increased affinity compared to the binding of the unmodified PD-L2 to the ectodomain of PD-1 and the ectodomain of RGMb.

27. The variant PD-L2 polypeptide of any of embodiments 1-26, wherein the variant PD-L2 polypeptide specifically binds to the ectodomain of PD-1 with increased affinity and specifically binds to the ectodomain of the other of RGMb with decreased affinity compared to the binding of the unmodified PD-L2 to the ectodomain of the other of RGMb.

28. The variant PD-L2 polypeptide of any of embodiments 24-27, wherein the increased affinity to the ectodomain of PD-1 is increased more than 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold or 60-fold compared to the unmodified PD-L2.

29. The variant PD-L2 polypeptide of embodiment 24 or embodiment 26, wherein the increased affinity to the ectodomain of RGMb is increased more than 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold or 60-fold compared to the unmodified PD-L2.

30. The variant PD-L2 polypeptide of embodiment 27, wherein the decreased affinity to the ectodomain of RGMb is decreased more than 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold or 60-fold compared to the unmodified PD-L2.

31. The variant PD-L2 polypeptide of any of embodiments 1-30, wherein the variant polypeptide specifically binds to the ectodomain of PD-1 with increased selectivity compared to the unmodified PD-L2.

32. The variant PD-L2 polypeptide of embodiment 31, wherein the increased selectivity comprises a greater ratio of binding of the variant polypeptide for PD-1 versus RGMb compared to the ratio of binding of the unmodified PD-L2 polypeptide for PD-1 versus RGMb.

33. The variant PD-L2 polypeptide of embodiment 32, wherein the ratio is greater by at least or at least about 1.5-fold, 2.0-fold, 3.0-fold, 4.0-fold, 5.0-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold or more.

34. The variant PD-L2 polypeptide of any of embodiments 9-33, wherein the PD-1 is a human PD-1.

35. The variant PD-L2 polypeptide of any of embodiments 9-34, wherein the RGMb is a human RGMb.

36. The variant PD-L2 polypeptide of any of embodiments 1-35, wherein the binding activity is altered (increased or decreased) more than 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold 40-fold or 50-fold compared to the unmodified PD-L2.

37. The variant PD-L2 polypeptide of any of embodiments 1-34 that is a soluble protein.

38. The variant PD-L2 polypeptide of any of embodiments 1-37, wherein:

the variant PD-L2 polypeptide lacks the PD-L2 transmembrane domain and intracellular signaling domain; and/or

the variant PD-L2 polypeptide is not capable of being expressed on the surface of a cell.

39. The variant PD-L2 polypeptide of any of embodiments 1-38, wherein the variant PD-L2 polypeptide is linked to a moiety that increases biological half-life of the polypeptide.

40. The variant PD-L2 polypeptide of any of embodiments 1-39, wherein the variant PD-L2 polypeptide is linked to a multimerization domain. 41. The variant PD-L2 polypeptide of embodiment 40, wherein the variant PD-L2 polypeptide is linked to an Fc domain or a variant Fc domain with reduced effector function.

42. The variant PD-L2 polypeptide of any of embodiments 39-41, wherein:

the Fc domain is mammalian, optionally human; or

the variant Fc domain comprises one or more amino acid modifications compared to an unmodified Fc domain that is mammalian, optionally human.

43. The variant PD-L2 polypeptide of any one of embodiments 41 and embodiment 42, wherein the Fc domain or variant thereof comprises the sequence of amino acids set forth in SEQ ID NO:211 or SEQ ID NO:212 or a sequence of amino acids that exhibits at least 85% sequence identity to SEQ ID NO:211 or SEQ ID NO:212.

44. The variant PD-L2 polypeptide of any of embodiments 41-43, wherein the Fc domain comprises one or more amino acid modifications selected from among E233P, L234A, L234V, L235A, L235E, G236del, G237A, S267K, N297G, V302C, and K447del each by EU numbering.

45. The variant PD-L2 polypeptide of any of embodiments 40-44, wherein the Fc domain comprises the amino acid modification C220S by EU numbering.

46. The variant PD-L2 polypeptide of any of embodiments 40-45, wherein the Fc domain comprises the sequence of amino acids set forth in any of SEQ ID NOS: 1189, 1205, 1206, 1207, 1739, 1738, 1739, 1740 or a sequence of amino acids that exhibits at least 85% sequence identity to any of SEQ ID NOS: 1189, 1205, 1206, 1207, 1739, 1738, 1739, 1740 and exhibits reduced effector function.

47. The variant PD-L2 polypeptide of any of embodiments 41-46, wherein the variant PD-L2 polypeptide is linked indirectly via a linker, optionally a G45 linker.

48. The variant PD-L2 polypeptide of any of embodiments 1-47, wherein the variant PD-L2 polypeptide is a transmembrane immunomodulatory protein further comprising a transmembrane domain, optionally wherein the transmembrane domain is linked, directly or indirectly, to the extracellular domain (ECD) or specific binding fragment thereof of the variant PD-L2 polypeptide.

49. The variant PD-L2 polypeptide of embodiment 48, wherein the transmembrane domain comprises the sequence of amino acids set forth as residues 221-241 of SEQ ID NO:4 or a functional variant thereof that exhibits at least 85% sequence identity to residues 221-241 of SEQ ID NO:4.

50. The variant PD-L2 polypeptide of embodiment 48 or embodiment 49, further comprising a cytoplasmic signaling domain, optionally wherein the cytoplasmic domain is linked, directly or indirectly, to the transmembrane domain.

51. The variant PD-L2 polypeptide of embodiment 50, wherein the cytoplasmic signaling domain comprises the sequence of amino acids set forth as residues 242-273 of SEQ ID NO:4 or a functional variant thereof that exhibits at least 85% sequence identity to residues 242-273 of SEQ ID NO:4.

52. The variant PD-L2 polypeptide of any of embodiments 1-51, wherein the variant PD-L2 increases IFN-gamma (interferon-gamma) expression relative to the unmodified PD-L2 in an in vitro T-cell assay.

53. The variant PD-L2 polypeptide of any of embodiments 1-51, wherein the variant PD-L2 decreases IFN-gamma (interferon-gamma) expression relative to the unmodified PD-L2 in an in vitro T-cell assay.

54. The variant PD-L2 polypeptide of any of embodiments 1-53 that is deglycosylated.

55. An immunomodulatory protein, comprising the variant PD-L2 polypeptide of any of embodiments 1-54 linked, directly or indirectly via a linker, to a second polypeptide comprising an immunoglobulin superfamily (IgSF) domain of an IgSF family member.

56. The immunomodulatory protein of embodiment 55, wherein the IgSF domain is an affinity-modified IgSF domain, said affinity-modified IgSF domain comprising one or more amino acid modifications compared to the unmodified or wild-type IgSF domain of the IgSF family member.

57. The immunomodulatory protein of embodiment 56, wherein the affinity modified IgSF domain exhibits altered binding to one or more of its cognate binding partner(s) compared to the binding of the unmodified or wild-type IgSF domain of the IgSF family member to the same one or more cognate binding partner(s).

58. The immunomodulatory protein of embodiment 57, wherein the IgSF domain exhibits increased binding to one or more of its cognate binding partner(s) compared to the binding of the unmodified or wild-type IgSF domain to the same one or more cognate binding partner(s).

59. The immunomodulatory polypeptide of any one of embodiments 56-58, wherein the variant PD-L2 is a first PD-L2 variant and the IgSF domain of the second polypeptide is an IgSF domain from a second variant PD-L2 of any of embodiments 1-58, wherein the first and second PD-L2 variant are the same or different.

60. The immunomodulatory protein of any one of embodiments 55-59, wherein the variant PD-L2 polypeptide is capable of specifically binding to PD-1 or RGMb and the IgSF domain of the second polypeptide is capable of binding to a cognate binding partner other than one specifically bound by the PD-L2 variant polypeptide.

61. The immunomodulatory protein of any of embodiments 55-60, wherein the IgSF domain is from a member of the B7 family.

62. The immunomodulatory protein of any of embodiments 55-60, wherein the IgSF domain is a tumor-localizing moiety that binds to a ligand expressed on a tumor or is an inflammatory-localizing moiety that binds to a ligand expressed on a cell or tissue of an inflammatory environment.

63. The immunomodulatory protein of embodiment 62, wherein the ligand is B7H6.

64. The immunomodulatory protein of embodiment 62 or embodiment 63, wherein the IgSF domain is from NKp30.

65. The immunomodulatory protein of any of embodiments 55-64, wherein the IgSF domain or affinity-modified IgSF domain thereof, optionally of the second or third polypeptide, is or comprises an IgV domain.

66. The immunomodulatory protein of any of embodiments 55-65, wherein the variant PD-L2 polypeptide is or comprises an IgV domain.

67. The immunomodulatory protein of any of embodiments 55-66, wherein the immunomodulatory protein further comprises a multimerization domain linked to at least one of the variant PD-L2 polypeptide or the second polypeptide, optionally wherein the multimerization domain is an Fc domain or a variant thereof with reduced effector function.

68. The immunomodulatory protein of any embodiments 55-67, wherein the IgSF domain of the second polypeptide is an IgSF domain of a ligand that binds to an inhibitory receptor, or is an affinity-modified IgSF domain thereof, optionally wherein the affinity-modified IgSF domain exhibits increased binding affinity and/or binding selectivity for the inhibitory receptor compared to binding of the unmodified IgSF domain to the same inhibitory receptor.

69. The immunomodulatory protein of embodiment 68, wherein:

the inhibitory receptor is TIGIT or CTLA-4; or

the ligand of the inhibitory receptor is CD155, CD112, or CD80.

70. The immunomodulatory protein of any of embodiments 55-69, wherein the second polypeptide is selected from:

(i) a wildtype CD155 comprising an IgSF set forth in any of SEQ ID NOS: 47, 344, or 387, or a variant CD155 polypeptide comprising an IgSF domain set forth in any of SEQ ID NOS: 345-386, 388-699, 1527-1736;

(ii) a wildtype CD112 comprising an IgSF domain set forth in any of SEQ ID NOS: 48, 700, or 795, or a variant CD112 polypeptide comprising an IgSF domain set forth in any of SEQ ID NOS: 701-794, 796-965, 1455-1526;

(iii) a wildtype CD80 comprising an IgSF set forth in any of SEQ ID NOS:28, 1039, or 2039, or a variant CD80 polypeptide comprising an IgSF domain set forth in any of SEQ ID NOS: 966-998, 1000-1038, 1040-1072, 1074-1112, 1114-1146, 1147-1186;

(iv) a wildtype PD-L1 comprising an IgSF set forth in any of SEQ ID NOS: 30, 1812, 1258, or 1454, or a variant PD-L1 polypeptide comprising an IgSF domain set forth in any of SEQ ID NOS:1259-1453, 1743-1811, 1813-2021;

(v) a sequence of amino acids that exhibits at least 95% sequence identity to any of the SEQ ID NOS in (i)-(iv) and that comprises the amino acid modifications, optionally amino acid substitutions, insertions and/or deletions thereof; or

(vi) a specific binding fragment of any of (i)-(v).

71. The immunomodulatory protein of any of embodiments 55-70, further comprising a third polypeptide comprising a wild-type IgSF domain or a variant or affinity-modified IgSF domain thereof, said affinity-modified IgSF domain comprising one or more amino acid modifications compared to the unmodified or wild-type IgSF domain of the IgSF family member, wherein:

the third polypeptide is the same as the first and/or second polypeptide; or

the third polypeptide is different from the first and/or second polypeptide.

72. The immunomodulatory protein of embodiment 71, wherein the IgSF domain of the third polypeptide is an affinity-modified IgSF domain comprising:

(i) a wildtype CD155 comprising an IgSF set forth in any of SEQ ID NOS: 47, 344, or 387, or a variant CD155 polypeptide comprising an IgSF domain set forth in any of SEQ ID NOS: 345-386, 388-699, 1527-1736;

(ii) a wildtype CD112 comprising an IgSF domain set forth in any of SEQ ID NOS: 48, 700, or 795, or a variant CD112 polypeptide comprising an IgSF domain set forth in any of SEQ ID NOS: 701-794, 796-965, 1455-1526;

(iii) a wildtype CD80 comprising an IgSF set forth in any of SEQ ID NOS:28, 1039, or 2039, or a variant CD80 polypeptide comprising an IgSF domain set forth in any of SEQ ID NOS: 966-998, 1000-1038, 1040-1072, 1074-1112, 1114-1146, 1147-1186;

(iv) a wildtype PD-L1 comprising an IgSF set forth in any of SEQ ID NOS: 30, 1812, 1258, or 1454, or a variant PD-L1 polypeptide comprising an IgSF domain set forth in any of SEQ ID NOS:1259-1453, 1743-1811, 1813-2021;

(v) a sequence of amino acids that exhibits at least 95% sequence identity to any of the SEQ ID NOS in (i)-(iv) and that comprises the amino acid modifications, optionally amino acid substitutions, insertions and/or deletions thereof; or

(vi) a specific binding fragment of any of (i)-(v).

73. The immunomodulatory protein of 70-72, further comprising at least one additional polypeptide comprising an IgSF domain of an IgSF family member or an affinity-modified IgSF domain thereof, said affinity-modified IgSF domain comprising one or more amino acid modifications compared to the binding of the unmodified or wild-type IgSF domain of the IgSF family member.

74. The immunomodulatory protein of any of embodiments 71-73, wherein the immunomodulatory protein further comprises a multimerization domain linked to at least one of the variant PD-L2 polypeptide, the second polypeptide and/or the third polypeptide, optionally wherein the multimerization domain is an Fc domain or a variant thereof with reduced effector function.

75. The immunomodulatory protein of any of embodiments 67-74, wherein the multimerization domain promotes heterodimer formation.

76. An immunomodulatory protein comprising a first variant PD-L2 polypeptide of any of embodiments 40-47 in which the multimerization domain is a first multimerization domain and a second variant PD-L2 polypeptide of any of embodiments 40-47 in which the multimerization domain is a second multimerization domain, wherein the first and second multimerization domains interact to form a multimer comprising the first and second variant PD-L2 polypeptide, optionally wherein the first and second variant PD-L2 polypeptides are the same.

77. An immunomodulatory protein comprising the immunomodulatory protein of any of embodiments 67-75, wherein the multimerization domain is a first multimerization domain and interacts with a second multimerization domain to form a multimer comprising the immunomodulatory protein.

78. The immunomodulatory protein of embodiment 77, wherein the immunomodulatory protein is a first immunomodulatory protein and a second immunomodulatory protein is linked directly or indirectly via a linker to the second multimerization domain, wherein the multimer comprises the first and second immunomodulatory protein.

79. The immunomodulatory protein of embodiment 78, wherein the second immunomodulatory protein is an immunomodulatory protein of any of caims 67-75, wherein the multimerization domain is the second multimerization domain.

80. The immunomodulatory protein of any of embodiments 76-79, wherein the multimer is a dimer.

81. The immunomodulatory protein of any of embodiments 76-80 that is a homodimer, optionally wherein the first and second multimerization domain is the same.

82. The immunomodulatory protein of any of embodiments 77-81, wherein the second polypeptide is a variant CD155 polypeptide and the first and/or second immunomodulatory protein comprises the sequence set forth in any of SEQ ID NOS: 1191-1196, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to any of SEQ ID NOS: 1191-1196.

83. The immunomodulatory protein of any of embodiments 76-80 that is a heterodimer, optionally wherein the first and second multimerization domain are different and/or are capable of interacting to mediate heterodimer formation.

84. The immunomodulatory protein of any of embodiments 77-80 and 83, wherein the second polypeptide is a variant CD155 polypeptide and:

the first or second immunomodulatory protein comprises the sequence set forth in any of SEQ ID NOS: 1197, 1198, 1199, 1200, 1201, 1203 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to any of SEQ ID NOS: 1197, 1198, 1199, 1200, 1201 or 1203; and

the other of the first or second immunomodulatory protein comprises the sequence set forth in any of SEQ ID NOS: 1188, 1190, 1202 or 1204, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to any of SEQ ID NOS: 1188, 1190, 1202 or 1204.

85. The immunomodulatory protein of any of embodiments 76-84, wherein the first and/or second multimerization domain is an Fc domain or a variant thereof with reduced effector function, optionally wherein:

the Fc domain is of an immunoglobulin protein that is human and/or the Fc region is human, optionally wherein the Fc region is of an immunoglobulin G1 (IgG1) or an immunoglobulin G2 (IgG2), optionally set forth in SEQ ID NO:211 or SEQ ID NO:212; or the variant Fc domain comprises one or more amino acid substitutions in a wildtype Fc region, optionally wherein the reduced effector function is reduced compared to a wildtype Fc region, optionally wherein the wildtype human Fc is of human IgG1.

86. The immunomodulatory protein of any of embodiments 76-85, wherein the first and second multimerization domain is the same or different.

87. The immunomodulatory protein of any of embodiments 65-75, 85 and 86, wherein the variant Fc region comprises:

the amino acid substitutions E233P, L234A, L234V, L235A, L235E, G236del, G237A, S267K, or N297G, with residue numbering according to the EU index of Kabat; or

the amino acid substitutions R292C/N297G/V302C or L234A/L235E/G237A, with residue numbering according to the EU index of Kabat.

88. The immunomodulatory protein of any of embodiments 65-75 and 85-87, wherein the Fc region or variant Fc region comprises the amino acid substitution C220S, with residue numbering according to the EU index of Kabat.

89. The immunomodulatory protein of any of embodiments 65-75 and 85-88, wherein the Fc region or variant Fc region comprises K447del, with residue numbering according to the EU index of Kabat.

90. A conjugate, comprising a variant PD-L2 of any of embodiments 1-54 or an immunomodulatory protein of any of embodiments 55-89 linked to a moiety.

91. The conjugate of embodiment 90, wherein the moiety is a targeting moiety that specifically binds to a molecule on the surface of a cell.

92. The conjugate of embodiment 91, wherein the targeting moiety specifically binds to a molecule on the surface of an immune cell, optionally wherein the immune cell is an antigen presenting cell or a lymphocyte.

93. The conjugate of embodiment 91, wherein the targeting moiety is a tumor-localizing moiety that binds to a molecule on the surface of a tumor.

94. The conjugate of any of embodiments 90-93, wherein the moiety is a protein, a peptide, nucleic acid, small molecule or nanoparticle.

95. The conjugate of any of embodiments 90-94, wherein the moiety is an antibody or antigen-binding fragment.

96. The conjugate of any of embodiments 90-95, wherein the conjugate is divalent, tetravalent, hexavalent or octavalent.

97. The conjugate of any of embodiments 90-96 that is a fusion protein.

98. A nucleic acid molecule(s), encoding a variant PD-L2 polypeptide of any of embodiments 1-59, an immunomodulatory protein of any of embodiments 55-89 or a conjugate that is a fusion protein of any of embodiments 90-97.

99. The nucleic acid molecule of embodiment 98 that is a synthetic nucleic acid.

100. The nucleic acid molecule of embodiment 98 or embodiment 99 that is cDNA.

101. A vector, comprising the nucleic acid molecule of any of embodiments 98-100.

102. The vector of embodiment 101 that is an expression vector.

103. The vector of embodiment 101 or embodiment 102, wherein the vector is a mammalian expression vector or a viral vector.

104. A cell, comprising the vector of any of embodiments 101-103.

105. The cell of embodiment 104 that is a mammalian cell.

106. The cell of embodiment 104 or embodiment 105 that is a human cell.

107. A method of producing a variant PD-L2 polypeptide or an immunomodulatory protein, comprising introducing the nucleic acid molecule of any of embodiments 98-100 or vector of any of embodiments 101-103 into a host cell under conditions to express the protein in the cell.

108. The method of embodiment 107, further comprising isolating or purifying the variant PD-L2 polypeptide or immunomodulatory protein from the cell.

109. A method of engineering a cell expressing a variant PD-L2 variant polypeptide, comprising introducing a nucleic acid molecule encoding the variant PD-L2 polypeptide of any of embodiments 1-54 into a host cell under conditions in which the polypeptide is expressed in the cell.

110. An engineered cell, expressing the variant PD-L2 polypeptide of any of embodiments 1-54, the immunomodulatory protein of any of embodiments 55-89, a conjugate that is a fusion protein of any of embodiments 90-97, the nucleic acid molecule of any of embodiments 98-100 or the vector of any of embodiments 101-102.

111. The engineered cell of embodiment 110, wherein the variant PD-L2 polypeptide or immunomodulatory protein is encoded by a nucleic acid comprising a sequence of nucleotides encoding a signal peptide.

112. The engineered cell of embodiment 110 or embodiment 111, wherein the variant PD-L2 polypeptide or immunomodulatory protein does not comprise a transmembrane domain and/or is not expressed on the surface of the cell.

113. The engineered cell of any of embodiments 110-112, wherein the variant PD-L2 polypeptide or immunomodulatory protein is secreted or is capable of being secreted from the engineered cell.

114. The engineered cell of embodiment 110 or embodiment 111, wherein the engineered cell comprises a variant PD-L2 polypeptide that comprises a transmembrane domain and/or is the transmembrane immunomodulatory protein of any of embodiments 48-54.

115. The engineered cell of embodiment 110, embodiment 111 or embodiment 114, wherein the variant PD-L2 polypeptide is expressed on the surface of the cell.

116. The engineered cell of any of embodiments 110-115, wherein the cell is an immune cell.

117. The engineered cell of embodiment 116, wherein the immune cell is an antigen presenting cell (APC) or a lymphocyte.

118. The engineered cell of any of embodiments 110-117, wherein the cell is a lymphocyte and the lymphocyte is a T cell.

119. The engineered cell of embodiment 118, wherein the cell is an APC and the APC is an artificial APC.

120. The engineered cell of any of embodiments 110-119 that is a primary cell.

121. The engineered cell of any of embodiments 110-120, wherein the cell is a mammalian cell.

122. The engineered cell of any of embodiments 110-121, wherein the cell is a human cell.

123. The engineered cell of any of embodiments 110-122, wherein the cell further comprises a chimeric antigen receptor (CAR) or an engineered T-cell receptor.

124. An infectious agent, comprising a nucleic acid molecule encoding a variant PD-L2 polypeptide of any of embodiments 1-54, an immunomodulatory polypeptide of any of embodiments 55-89 or a conjugate that is a fusion protein of any of claims 90-97.

125. The infectious agent of embodiment 124, wherein the encoded variant PD-L2 polypeptide or immunomodulatory polypeptide does not comprise a transmembrane domain and/or is not expressed on the surface of a cell in which it is expressed.

126. The infectious agent of embodiment 124 or embodiment 125, wherein the encoded variant PD-L2 polypeptide or immunomodulatory polypeptide is secreted or is capable of being secreted from a cell in which it is expressed.

127. The infectious agent of embodiment 124, wherein the encoded variant PD-L2 polypeptide comprises a transmembrane domain.

128. The infectious agent of embodiment 124 or embodiment 127, wherein the encoded variant PD-L2 polypeptide is expressed on the surface of a cell in which it is expressed.

129. The infectious agent of any of embodiments 124-128, wherein the infectious agent is a bacterium or a virus.

130. The infectious agent of embodiment 129, wherein the infectious agent is a virus and the virus is an oncolytic virus.

131. The infectious agent of embodiment 130, wherein the oncolytic virus is an adenovirus, adeno-associated virus, herpes virus, Herpes Simplex Virus, Vesticular Stomatic virus, Reovirus, Newcastle Disease virus, parvovirus, measles virus, vesticular stomatitis virus (VSV), Coxsackie virus or a Vaccinia virus.

132. The infectious agent of embodiment 130 or embodiment 131, wherein the virus specifically targets dendritic cells (DCs) and/or is dendritic cell-tropic.

133. The infectious agent of embodiment 132, wherein the virus is a lentiviral vector that is pseudotyped with a modified Sindbis virus envelope product.

134. The infectious agent of any of embodiments 124-133, further comprising a nucleic acid molecule encoding a further gene product that results in death of a target cell or that can augment or boost an immune response.

135. The infectious agent of embodiment 134, wherein the further gene product is selected from an anticancer agent, anti-metastatic agent, an antiangiogenic agent, an immunomodulatory molecule, an immune checkpoint inhibitor, an antibody, a cytokine, a growth factor, an antigen, a cytotoxic gene product, a pro-apoptotic gene product, an anti-apoptotic gene product, a cell matrix degradative gene, genes for tissue regeneration or a reprogramming human somatic cells to pluripotency.

136. A pharmaceutical composition, comprising the variant PD-L2 polypeptide of any of embodiments 1-54, an immunomodulatory protein of any of embodiments 55-89, a conjugate of any of embodiments 90-97 or an engineered cell of any of embodiments 110-123 or an infectious agent of any of embodiments 124-135.

137. The pharmaceutical composition of embodiment 136, comprising a pharmaceutically acceptable excipient.

138. The pharmaceutical composition of embodiment 136 or 137, wherein the pharmaceutical composition is sterile.

139. An article of manufacture comprising the pharmaceutical composition of any of embodiments 136-138 in a vial.

140. The article of manufacture of embodiment 139, wherein the vial is sealed. 141. A kit comprising the pharmaceutical composition of any of embodiments 136-138, and instructions for use.

142. A kit comprising the article of manufacture according to embodiment 139 and 140, and instructions for use.

143. A method of modulating an immune response in a subject, comprising administering the pharmaceutical composition of any of embodiments 136-138 to the subject.

144. A method of modulating an immune response in a subject, comprising administering the engineered cells of any of embodiments 110-123.

145. The method of embodiment 144, wherein the engineered cells are autologous to the subject.

146. The method of embodiment 144, wherein the engineered cells are allogenic to the subject.

147. The method of any of embodiments 143-146, wherein modulating the immune response treats a disease or condition in the subject.

148. The method of any of embodiments 143-147, wherein the immune response is increased.

149. The method of any of embodiments 143-148, wherein a variant PD-L2 polypeptide or immunomodulatory protein that is soluble, optionally that lacks a PD-L2 transmembrane and intracellular signaling domain, is administered to the subject.

150. The method of any of embodiments 143-149, wherein the variant polypeptide or immunomodulatory protein is an Fc fusion protein.

151. The method of any of embodiments 143 and 148-150, wherein a variant PD-L2 polypeptide of any of embodiments 1-54, or the immunomodulatory protein of any of embodiments 55-89 is administered to the subject.

152. The method of any of embodiments 143-149 wherein an engineered cell comprising a secretable variant PD-L2 polypeptide is administered to the subject.

153. The method of any of embodiments 143 and 148-152, wherein an engineered cell of any of embodiments 98-101 and 104-111 is administered to the subject.

154. The method of any of embodiments 143, 147 and 148, wherein an infectious agent encoding a variant PD-L2 polypeptide that is a secretable immunomodulatory protein is administered to the subject, optionally under conditions in which the infectious agent infects a tumor cell or immune cell and the secretable immunomodulatory protein is secreted from the infected cell.

155. The method of any of embodiments 143-154, wherein the disease or condition is a tumor or cancer.

156. The method of any one of embodiments 143-155, wherein the disease or condition is selected from melanoma, lung cancer, bladder cancer, a hematological malignancy, liver cancer, brain cancer, renal cancer, breast cancer, pancreatic cancer, colorectal cancer, spleen cancer, prostate cancer, testicular cancer, ovarian cancer, uterine cancer, gastric carcinoma, a musculoskeletal cancer, a head and neck cancer, a gastrointestinal cancer, a germ cell cancer, or an endocrine and neuroendocrine cancer.

157. The method of any of embodiments 143-147, wherein the immune response is decreased.

158. The method of any of embodiments 143-147 and 157, wherein an immunomodulatory protein or conjugate comprising a variant PD-L2 polypeptide linked to an IgSF domain or a moiety that localizes to a cell or tissue of an inflammatory environment is administered to the subject.

159. The method of embodiment 158, wherein the binding molecule comprises an antibody or an antigen-binding fragment thereof or comprises a wild-type IgSF domain or variant thereof.

160. The method of any of embodiments 143-147 and 157-159, wherein the immunomodulatory protein of any of embodiments 57-77 or the conjugate of any of embodiments 90-97 is administered to the subject.

161. The method of any of embodiments 143-147 and 157, wherein a variant PD-L2 polypeptide that is a transmembrane immunomodulatory protein is administered to the subject.

162. The method of any of embodiments 144-147 and 157-161, wherein the engineered cell comprising a variant PD-L2 polypeptide that is a transmembrane immunomodulatory protein of any of embodiments 50-59 is administered to the subject.

163. The method of any of embodiments 143, 147 and 157, wherein an infectious agent encoding a variant PD-L2 polypeptide that is a transmembrane immunomodulatory protein is administered to the subject, optionally under conditions in which the infectious agent infects a tumor cell or immune cell and the transmembrane immunomodulatory protein is expressed on the surface of the infected cell.

164. The method of any of embodiments 143-147 and 157-163, wherein the disease or condition is an inflammatory or autoimmune disease or condition.

165. The method of any of embodiments 143-147 and 157-164 wherein the disease or condition is an Antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis, a vasculitis, an autoimmune skin disease, transplantation, a Rheumatic disease, an inflammatory gastrointestinal disease, an inflammatory eye disease, an inflammatory neurological disease, an inflammatory pulmonary disease, an inflammatory endocrine disease, or an autoimmune hematological disease.

166. The method of embodiment 164 or embodiment 165, wherein the disease or condition is selected from inflammatory bowel disease, transplant, Crohn's disease, ulcerative colitis, multiple sclerosis, asthma, rheumatoid arthritis, or psoriasis.

IX. EXAMPLES

The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

Example 1 Generation of Mutant DNA Constructs of IgSF Domains

Example 1 describes the generation of mutant DNA constructs of human PD-L2 IgSF domains for translation and expression on the surface of yeast as yeast display libraries.

A. Degenerate Libraries

Constructs were generated based on a wildtype human PD-L2 sequence set forth in SEQ ID NO:115 containing the immunoglobulin-like V-type (IgV) domain as follows:

-   -   LFTVTVPKELYIIEHGSNVTLECNFDTGSHVNLGAITASLQKVENDTSPHRERATLLEE         QLPLGKASFHIPQVQVRDEGQYQCIIIYGVAWDYKYLTLKVKA (SEQ ID NO:115)

For libraries that target specific residues for complete or partial randomization with degenerate codons, degenerate codons, such as specific mixed base sets to code for various amino acid substitutions, are generated using an algorithm at the URL:rosettadesign.med.unc.edu/SwiftLib/. In general, positions to mutate were chosen from direct crystal structure information for PDL2::PD1 complex (e.g. PDB: 3BP5). Alternatively, a homology model may be generated if structures of homologous protein complexes are unavailable. Using the structural information, a structure viewer (available at the URL: spdbv.vital-it.ch) can be used to identify contact or non-contact interface residues for mutagenesis with degenerate codons

The next step in library design can be the alignment of human, mouse, rat, and monkey PD-L2 sequences to identify which of the residues chosen for mutagenesis are conserved residues. Based on this analysis, conserved target residues can be mutated with degenerate codons that only specify conservative amino acid changes plus the wild-type residue. Residues that are not conserved can be mutated more aggressively, but also included the wild-type residue. Degenerate codons that also encoded the wild-type residue are deployed to avoid excessive mutagenesis of target protein. For the same reason, only up to 20 positions are targeted for mutagenesis for each library. Selection of residues for mutagenesis is focused on contact, such as target side chain residues that interact with the ligand, and non-contact interfacial residues that are within 6 Å of the binding surface with their side chains directed toward the ligand/counter structure.

To generate DNA encoding the targeted library, overlapping oligos of up to 80 nucleotides in length and containing degenerate codons at the residue positions targeted for mutagenesis, can be ordered from Integrated DNA Technologies (Coralville, USA). The oligonucleotides are dissolved in sterile water, mixed in equimolar ratios, heated to 95° C. for five minutes and slowly cooled to room temperature for annealing. IgV domain-specific oligonucleotide primers that anneal to the start and end of the IgV domain gene sequence are then used to generate a first PCR product. IgV domain-specific oligonucleotides which overlap by 40 bp with pBYDS03 cloning vector (Life Technologies USA), beyond and including the BamHI and KpnI cloning sites, are then used to amplify 100 ng of PCR product from the prior step to generate a total of at least 12 μg of DNA for every electroporation. Both PCR's were by polymerase chain reaction (PCR) using OneTaq 2×PCR Master Mix (New England Biolabs, USA). The second PCR products are purified using a PCR purification kit (Qiagen, Germany) and resuspended in sterile deionized water. Alternatively, Ultramers (Integrated DNA Technologies) of up to 200 bp in length can be used in conjunction with megaprimer PCR (URL: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC146891/pdf/253371.pdf) to generate larger stretches of degenerate codons that could not be as easily generated using multiple small overlapping primers. Following the generation of full length product using megaprimer PCR, the mutant IgV domain library is PCR amplified again using DNA primers containing 40 bp overlap region with the pBYDS03 cloning vector for homologous recombination into yeast.

To prepare for library insertion, pBYDS03 vector is digested with BamHI and KpnI restriction enzymes (New England Biolabs, USA) and the large vector fragment is gel-purified and dissolved in sterile, deionized water. Electroporation-ready DNA for the next step is generated by mixing 12 μg of library DNA insert with 4 μg of linearized vector in a total volume of 50 μL deionized and sterile water. An alternative method to generate targeted libraries, is to carry out site-directed mutagenesis (Multisite kit, Agilent, USA) of the target IgV domain with oligonucleotides containing degenerate codons. This approach can be used to generate sublibraries that only target a few specific stretches of DNA for mutagenesis. In these cases, sublibraries can be mixed before proceeding to the selection steps. In general, library sizes are in the range of 10E7 to 10E8 clones, except that sublibraries are only in the range of 10E4 to 10E5.

B. Random Libraries

Random libraries were also constructed to identify variants of the IgV domain of PD-L2 set forth in SEQ ID NO:115. DNA encoding the wild-type IgV domain was cloned between the BamHI and KpnI sites of the yeast display vector pBYDS03. The DNA was then mutagenized with the Genemorph II Kit (Agilent, USA) to generate an average of three to five amino acid changes per library variant. Mutagenized DNA was then amplified by the two-step PCR and further processed as described above for targeted libraries.

Example 2 Introduction of DNA Libraries into Yeast

Example 2 describes the introduction of PD-L2 DNA libraries into yeast.

To introduce library DNA into yeast, electroporation-competent cells of yeast strain BJ5464 (ATCC.org; ATCC number 208288) were prepared and electroporated on a Gene Pulser II (Biorad, USA) with the electroporation-ready DNA from the steps above essentially as described (Colby, D. W. et al. 2004 Methods Enzymology 388, 348-358). The only exception is that transformed cells were grown in non-inducing minimal selective SCD-Leu medium to accommodate the LEU2 selectable marker carried by modified plasmid pBYDS03. One liter of SCD-Leu media consists of 14.7 grams sodium citrate, 4.29 grams citric acid monohydrate, 20 grams dextrose, 6.7 grams yeast nitrogen base, and 1.6 grams yeast synthetic drop-out media supplement without leucine. The Medium was filter sterilized before use using a 0.22 vacuum filter device.

Library size was determined by plating serial dilutions of freshly recovered cells on SCD-Leu agar plates and then extrapolating library size from the number of single colonies from plating that generated at least 50 colonies per plate. In general, library sizes ranged from 10E8 to 10E9 transformants based on this dilution assay. The remainder of the electroporated culture was grown to saturation in SCD-Leu and cells from this culture were subcultured 1/100 into fresh SCD-Leu once more and grown to saturation to minimize the fraction of untransformed cells, and grown overnight and to allow for segregation of plasmid from cells that may contain two or more library variants. To maintain library diversity, this subculturing step was carried out using an inoculum that contained at least 10× more cells than the calculated library size. Cells from the second saturated culture were resuspended in fresh medium containing sterile 25% (weight/volume) glycerol to a density of 10E10/mL and frozen and stored at −80° C. (frozen library stock).

Example 3 Yeast Selection

Example 3 describes the selection of yeast expressing affinity modified variants of PD-L2.

A number of cells equal to at least 10 times the estimated library size were thawed from individual library stocks, suspended to 0.1×10E6 cells/mL in non-inducing SCD-Leu medium, and grown overnight. The next day, a number of cells equal to 10 times the library size were centrifuged at 2000 RPM for two minutes and resuspended to 0.5×10E6 cells/mL in inducing SCDG-Leu media. One liter of the SCDG-Leu induction media consists of 5.4 grams Na₂HPO₄, 8.56 grams of NaH₂PO₄—H₂O, 20 grams galactose, 2.0 grams dextrose, 6.7 grams yeast nitrogen base, and 1.6 grams of yeast synthetic drop out media supplement without leucine dissolved in water and sterilized through a 0.22 μm membrane filter device. The culture was grown in induction medium for 1 day at room temperature to induce expression of library proteins on the yeast cell surface.

Cells were sorted two to three times using magnetic beads loaded with cognate ligand to reduce non-binders and enrich for all variant PD-L2 with the ability to bind their exogenous recombinant counter-structure proteins. This was then followed by one to two rounds of fluorescence activated cell sorting (FACS) using decreasing concentrations of exogenous counter-structure protein staining in each round to enrich the fraction of yeast cells that displays improved binders. Magnetic bead enrichment and selections by flow cytometry were carried out essentially as described in Miller, K. D. et al., Current Protocols in Cytometry 4.7.1-4.7.30, July 2008.

With PD-L2 libraries, target ligand proteins was rhPD-1.Fc (i.e., recombinant human PD-1-Fc fusion protein from R&D Systems, USA). Magnetic Protein A beads were obtained from New England Biolabs, USA. For two-color, flow cytometric sorting, a Bio-Rad S3e sorter was used. PD-L2 display levels were monitored with an anti-hemagglutinin antibody labeled with Alexafluor 488 (Life Technologies, USA). Ligand binding of Fc fusion proteins to rhPD-1.Fc was detected with rPhycoerythrin (PE) conjugated anti-human Ig specific goat Fab (Jackson ImmunoResearch, USA). Doublet yeast were gated out using forward scatter (FSC)/side scatter (SSC) parameters, and sort gates were based upon higher ligand binding detected in FL2 that possessed more limited tag expression binding in FL1.

Yeast outputs from the flow cytometric sorts were assayed for higher specific binding affinity. Sort output yeast were expanded and re-induced to express the particular IgSF affinity modified domain variants they encode. This population then can be compared to the parental, wild-type yeast strain, or any other selected outputs, such as the bead output yeast population, by flow cytometry.

For PD-L2, the second round FACS outputs (F2) were compared to parental for binding rPD-1.Fc by double staining each population with anti-HA (hemagglutinin) tag expression and the anti-human Fc-PE secondary to detect ligand binding.

Selected variant PD-L2 IgV domains were further formatted as fusion proteins and tested for binding and functional activity as described below.

Example 4 Reformatting Selection Outputs as Fc-Fusions and in Various Immunomodulatory Protein Types

Example 4 describes reformatting of selection outputs identified in Example 3 as immunomodulatory proteins containing an affinity modified (variant) immunoglobulin-like V-type (IgV) domain of PD-L2 fused to an Fc molecule (variant IgV domain-Fc fusion molecules).

Output cell pools from final flow cytometric PD-L2 sorts were grown to terminal density in SCD-Leu medium. Plasmid DNA from at least 10× the number of cells of each sort output was isolated using a yeast plasmid DNA isolation kit (Zymo Research, USA). For Fc fusions, PCR primers with added restriction sites suitable for cloning into the Fc fusion vector of choice were used to batch-amplify from the plasmid DNA preps the coding DNA for the mutant target IgV domains After restriction digestion, the PCR products were ligated into Fc fusion vector followed by heat shock transformation into E. coli strain XL1 Blue (Agilent, USA) or NEB5alpha (New England Biolabs) as directed by supplier. Alternatively, the outputs were PCR amplified with primers containing 40 bp overlap regions on either end with Fc fusion vector to carry out in vitro recombination using Gibson Assembly Mastermix (New England Biolabs, USA), which was subsequently used for heat shock transformation into E. coli strain NEB5alpha. Exemplary of an Fc fusion vector is pFUSE-hIgG1-Fc2 (InvivoGen, USA).

Dilutions of transformation reactions were plated on LB-agar containing 100 μg/mL carbenicillin (Teknova, USA) to isolate single colonies for selection. Up to 96 colonies from each transformation were then grown in 96 well plates to saturation overnight at 37° C. in LB-broth (Teknova cat #L8112) and a small aliquot from each well was submitted for DNA sequencing of the IgV domain insert in order to identify mutation(s) in all clones. Sample preparation for DNA sequencing was carried out using protocols provided by the service provider (Genewiz; South Plainfield, N.J.). After removal of sample for DNA sequencing, glycerol was then added to the remaining cultures for a final glycerol content of 25% and plates were stored at −20° C. for future use as master plates (see below). Alternatively, samples for DNA sequencing were generated by replica plating from grown liquid cultures onto solid agar plates using a disposable 96 well replicator (VWR, USA). These plates were incubated overnight to generate growth patches and the plates were submitted to Genewiz as specified by Genewiz.

After analysis of the Genewiz-generated DNA sequencing data, clones of interest were recovered from master plates and individually grown to saturation in liquid LB-broth containing 100m/mL carbenicillin (Teknova, USA) and cultures were then used for preparation of plasmid DNA of each clone using a standard kit such as the PureYield Plasmid Miniprep System (Promega, USA) or the MidiPlus kit (Qiagen). Identification of clones of interest from Genewiz sequencing data generally involved the following steps. First, DNA sequence data files were downloaded from the Genewiz web site. All sequences were then manually curated so that they start at the beginning of the IgV domain coding region. The curated sequences were then batch-translated using a suitable program available at the URL: www.ebi.ac.uk/Tools/st/emboss transeq/. The translated sequences were then aligned using a suitable program available at the URL: multalin.toulouse.inra.fr/multalin/multalin.html. Alternatively, Genewiz sequenced were processed to generate alignments using Ugene software (http://ugene.net).

Clones of interest were then identified from alignments using the following criteria: 1.) identical clone occurs at least two times in the alignment and 2.) a mutation occurs at least two times in the alignment and preferably in distinct clones. Clones that meet at least one of these criteria were assumed to be clones that have been enriched by the sorting process most likely due to improved binding.

To generate recombinant immunomodulatory proteins that are Fc fusion proteins containing an IgV domain of PD-L2 with at least one affinity-modified domain (e.g. variant PD-L2 IgV-Fc), the encoding DNA was generated to encode a protein as follows: variant (mutant) PD-L2 IgV domain followed by a linker of three alanines (AAA) followed by a human IgG1 Fc set forth in SEQ ID NO:1205 containing the mutations R292C, N297G and V302C by EU numbering (corresponding to R77C, N82G and V87C with reference to wild-type human IgG1 Fc set forth in SEQ ID NO: 211). Since the construct does not include any antibody light chains that can form a covalent bond with a cysteine, the human IgG1 Fc also contains replacement of the cysteine residues to a serine residue at position 220 (C220S) by EU numbering (corresponding to position 5 (C5S) with reference to the wild-type or unmodified Fc set forth in SEQ ID NO: 211).

Example 5 Expression and Purification of Fc-Fusions

Example 5 describes the high throughput expression and purification of Fc-fusion proteins containing variant IgV PD-L2 as described in the above Examples.

Recombinant variant Fc fusion proteins were produced from suspension-adapted human embryonic kidney (HEK) 293 cells with Expi293 expression system (Invitrogen, USA). 4 μg of each plasmid DNA from the previous step was added to 200 μL Opti-MEM (Invitrogen, USA) at the same time as 10.84, ExpiFectamine was separately added to another 200 μL Opti-MEM. After 5 minutes, the 200 μL of plasmid DNA was mixed with the 200 μL of ExpiFectamine and was further incubated for an additional 20 minutes before adding this mixture to cells. Ten million Expi293 cells were dispensed into separate wells of a sterile 10 mL, conical bottom, deep 24 well growth plate (Thomson Instrument Company, USA) in a volume 4 mL Expi293 media (Invitrogen, USA). Plates were shaken for 5 days at 120 RPM in a mammalian cell culture incubator set to 95% humidity and 8% CO₂. Following a 5-day incubation, cells were pelleted and culture supernatants were retained.

Protein was purified from supernatants using a high throughput 96 well Filter Plate (Thomson Catalog number 931919), each well loaded with 604, of Mab SelectSure settled bead (GE Healthcare cat. no. 17543801). Protein was eluted with four consecutive 200 μL fractions of 50 mM Acetate pH3.3. Each fraction's pH was adjusted to above pH 5.0 with 44, 2M Tris pH 8.0. Fractions were pooled and quantitated using 280 nm absorbance measured by Nanodrop instrument (Thermo Fisher Scientific, USA), and protein purity was assessed by loading 5 μg of protein on NUPAGE pre-cast, polyacrylamide gels (Life Technologies, USA) under denaturing and non-reducing conditions and subsequent gel electrophoresis. Proteins were visualized in gel using standard Coomassie staining.

Example 6 Assessment of Binding of Affinity-Matured IgSF Domain-Containing Molecules

A. Binding to Cell-Expressed Counter Structures

This Example describes Fc-fusion binding studies of purified proteins from the above Examples to assess specificity and affinity of PD-L2 domain variant immunomodulatory proteins for the cognate binding partner.

Binding studies were carried out on transfected HEK293 cells generated to express the full-length mammalian surface ligands using Jurkat/IL-2 reporter cells (purchased from Promega Corp. USA) that were then transduced to stably express PD-1 (Jurkat/PD-1). For staining by flow cytometry, 100,000 Jurkat/PD-1 cells or negative control (Jurkat only) were plated in 96 well round bottom plates. Cells were spun down and resuspended in staining buffer (PBS (phosphate buffered saline), 1% BSA (bovine serum albumin), and 0.1% sodium azide) for 20 minutes to block non-specific binding. Afterwards, cells were centrifuged again and resuspended in 50 μL staining buffer containing 100 nM to 46 pM of each candidate PD-L2 variant Fc fusion protein. As controls, a full extracellular domain of wild-type PD-L2 (composed of one IgV and one IgC domain) fused to Fc (“Full length ECD of PD-L2”) and an IgV domain of wild-type PD-L2 (“wild type PD-L2 IgV”) or, in some cases, anti-PD-1 monoclonal antibody (nivolumab), were tested. Primary staining was performed on ice for 45 minutes, before washing cells twice in 150 μL staining buffer. PE-conjugated anti-human Fc (Jackson ImmunoResearch, USA) was diluted 1:150 in 50 μl staining buffer and added to cells and incubated another 30 minutes on ice. Secondary antibody was washed out twice, cells were fixed in 4% formaldehyde/PBS, and samples were analyzed on Intellicyt flow cytometer (Intellicyt Corp, USA).

Mean Fluorescence Intensity (MFI) was calculated and compared to wild type PD-L2 IgV with FlowJo Version 10 (FlowJo LLC, USA). Specifically, the binding activity as measured by the Mean Fluorescence Intensity (MFI) value for binding of 50 nM of each variant Fc-fusion molecule to Jurkat/PD-1 cells and the ratio of the MFI compared to the binding of the corresponding wild type (unmodified) PD-L2 IgV fusion molecule not containing the amino acid substitution(s) to PD-1 was determined.

B. ForteBio Octet Binding Assay

Protein-protein interactions between PD-1 and PD-L2 domain variant immunomodulatory proteins were further assessed using Fortebio binding assays. PD-1 was loaded individually onto anti-human capture sensors (ForteBio Octet AHC) and Fc fusions of full length wildtype (unmodified) PD-L2 IgV, full length ECD of PD-L2, full length ECD of PD-L1, or variant PD-L2 Fc-fusion molecules were bound to the PD-1 receptor at a single concentration of 100 nM. As a positive control, anti-PD-1 monoclonal antibody (nivolumab) was also assessed.

Loading response of anti-human capture sensors of each binding protein being tested with the variant IgV-Fc fusion molecule was determined.

C. Results

Results for the binding studies for exemplary tested variant PD-L2 IgV-Fc fusion molecules are shown in Table 10. Table 10 indicates amino acid substitutions in the IgV of the variant PD-L2 selected in the screen described above. In the Table and Figure, the exemplary amino acid substitutions and insertions in the IgV domain are designated by amino acid position number corresponding to amino acid positions in the respective reference unmodified PD-L2 extracellular domain (ECD) sequence set forth in SEQ ID NO:31. The amino acid position is indicated in the middle, with the corresponding unmodified (e.g. wild-type) amino acid listed before the number and the identified variant amino acid substitution (or inserted designated by “ins”) listed after the number. Column 2 sets forth the SEQ ID NO identifier for each variant IgV domain contained in the variant IgV-Fc fusion molecule.

As shown in Table 10, the selections resulted in the identification of a number of PD-L2 IgSF (e.g. IgV) domain variants that were affinity-modified to exhibit increased binding for PD-1 as determined by flow cytometry binding studies to Jurkat/PD-1 cells or by Fortebio binding analysis. FIG. 8 depicts the MFI for binding to Jurakt/PD-1 cells at various concentrations of PD-L2 variant Fc fusion protein. As shown in FIG. 8, the tested variants exhibit improved binding to Jurkat/PD-1 compared to PDL2-IgV, and to levels that were similar to anti-PD-1 monoclonal antibody (nivolumab).

TABLE 10 Variant PD-L2 selected against PD-1. Molecule sequence and binding data. Binding to Jurkat/ Fortebio PD-1 Cells binding SEQ Fold increase to PD-1- ID MFI over wildtype Fc NO at PD-L2 Response PD-L2 mutation(s) (IgV) 50 nM IgV-Fc Units H15Q 268 15998 1.63 0.007 N24D 269 1414 0.14 −0.039 E44D 270 2928 0.3 −0.006 V89D 271 3361 0.34 0.005 Q82R,V89D 272 44977 4.57 1.111 E59G,Q82R 273 12667 1.29 −0.028 S39I,V89D 274 26130 2.65 0.26 S67L,V89D 275 15991 1.62 0.608 S67L,I85F 276 529 0.05 −0.005 S67L,I86T 277 6833 0.69 0.141 H15Q,K65R 278 13497 1.37 −0.001 H15Q,Q72H,V89D 279 12629 1.28 0.718 H15Q,S67L,R76G 280 47201 4.8 0.418 H15Q,R76G,185F 281 2941 0.3 −0.038 H15Q,T47A,Q82R 282 65174 6.62 0.194 H15Q,Q82R,V89D 283 49652 5.04 1.198 H15Q,C23S,I86T 284 830 0.08 −0.026 H15Q,S39I,I86T 285 1027 0.1 0.309 H15Q,R76G,I85F 286 1894 0.19 −0.006 E44D,V89D,W91R 287 614 0.06 −0.048 I13V,S67L,V89D 288 26200 2.66 1.42 H15Q,S67L,I86T 289 15952 1.62 0.988 I13V,H15Q,S67L,I86T 290 21570 2.19 1.391 I13V,H15Q,E44D,V89D 291 23958 2.43 1.399 I13V,S39I,E44D,Q82R,V89D 292 71423 7.26 0.697 I13V,E44D,Q82R,V89D 293 45191 4.59 1.283 Il3V,Q72H,R76G,I86T 294 10429 1.06 0.733 Il3V,H15Q,R76G,I85F 295 4736 0.48 −0.04 H15Q,S67L,R76G,I85F 297 2869 0.29 0.025 H15Q,S39I,R76G,V89D 298 Little or no protein produced H15Q,T47A,Q72H,R76G,I86T 298 32103 3.26 0.512 H15Q,T47A,Q72H,R76G 299 16500 1.68 0.327 I13V,H15Q,T47A,Q72H,R76G 300 73412 7.46 0.896 H15Q,E44D,R76G,I85F 301 2885 0.29 −0.013 H15Q,S39I,S67L,V89D 302 45502 4.62 1.174 H15Q,N32D,S67L,V89D 303 25880 2.63 1.407 N32D,S67L,V89D 304 31753 3.23 1.155 H15Q,S67L,Q72H,R76G,V89D 305 40180 4.08 1.464 H15Q,Q72H,Q74R,R76G,I86T 306 4049 0.41 0.093 G28V,Q72H,R76G,I86T 307 5563 0.57 0.003 I13V,H15Q,S39I,E44D,S67L 308 63508 6.45 0.889 E44D,S67L,Q72H,Q82R,V89D 309 51467 5.23 1.061 H15Q,V89D 310 17672 1.8 0.31 H15Q,T47A 311 26578 2.7 0.016 I13V,H15Q,Q82R 312 76146 7.74 0.655 I13V,H15Q,V89D 313 28745 2.92 1.331 I13V,S67L,Q82R,V89D 314 58992 5.99 1.391 I13V,H15Q,Q82R,V89D 315 49523 5.03 1.419 H15Q,V31M,S67L,Q82R,V89D 316 67401 6.85 1.37 I13V,H15Q,T47A,Q82R 317 89126 9.05 0.652 I13V,H15Q,V31A,N45S,Q82R,V89D 318 68016 6.91 1.327 I13V, T20A, T47A, K65X*, Q82R, V89D 319 36743 3.73 0.092 H15Q,T47A,H69L,Q82R,V89D 320 65598 6.66 1.44 I13V,H15Q,T47A,H69L,R76G,V89D 321 54340 5.52 1.719 I12V,I13V,H15Q,T47A,Q82R,V89D 322 61207 6.22 1.453 I13V,H15Q,R76G,D77N,Q82R,V89D 323 33079 3.36 0.065 I13V,H15Q,T47A,R76G,V89D 324 53668 5.45 1.596 I13V,H15Q,T47A,Q82R,V89D 325 63320 6.43 1.418 I13V,H15Q,T47A,Q82R,V89D 325 60980 6.2 1.448 I13V,H15Q,I36V,T47A,S67L,V89D 327 52835 5.37 1.627 H15Q,T47A,K65R,S67L,Q82R,V89D 328 79692 8.1 1.453 H15Q,L33P,T47A,S67L,P71S,V89D 329 45726 4.65 1.467 I13V,H15Q,Q72H,R76G,I86T 330 24450 2.48 1.355 H15Q,T47A,S67L,Q82R,V89D 331 67962 6.9 1.479 F2L,H15Q,D46E,T47A,Q72H,R76G,Q82R,V89D 332 23039 2.34 1.045 I13V,H15Q,L33F,T47A,Q82R,V89D 333 62254 6.32 1.379 H15Q,N24S,T47A,Q72H,R76G,V89D 335 32077 3.26 0.4 I13V,H15Q,E44V,T47A,Q82R,V89D 336 61005 6.2 1.329 H15Q,N18D,T47A,Q72H,V73A,R76G,I86T,V89D 337 48317 4.91 0.475 I13V,H15Q,T37A,E44D,S48C,S67L,Q82R,V89D 338 47605 4.84 1.255 H15Q,L33H,S67L,R76G,Q82R,V89D 339 62326 6.33 1.507 I13V,H15Q,T47A,Q72H,R76G,I86T 340 49016 4.98 1.477 H15Q,S39I,E44D,Q72H,V75G,R76G,Q82R,V89D 341 43713 4.44 0.646 H15Q,T47A,S67L,R76G,Q82R,V89D 342 71897 7.3 1.539 I13V,H15Q,T47A,S67L,Q72H,R76G,Q82R,V89D 343 71755 7.29 1.536 Wild Type PD-L2 IgV 115 9843 1 −0.024 Full length ECD of PD-L2 31 2145 0.22 0.071 Full length ECD of PD-L1 (R&D Systems) 30 23769 2.41 1.263 Anti-PD-1 monoclonal antibody (nivolumab) — 87002 8.84 0.899 *stop codon at indicated position

Example 7 Assessment of Activity of Affinity-Matured IgSF Domain-Containing Molecules with Mixed Lymphocyte Reaction (MLR)

This Example describes Fc-fusion variant protein bioactivity characterization in human primary T cell in vitro assays.

Soluble variant PD-L2 IgV-Fc bioactivity was tested in a human Mixed Lymphocyte Reaction (MLR). Human primary dendritic cells (DC) were generated by culturing monocytes isolated from PBMC (BenTech Bio, USA) in vitro for 7 days with 50 ng/mL rIL-4 (R&D Systems, USA) and 80 ng/mL rGM-CSF (R&D Systems, USA) in Ex-Vivo 15 media (Lonza, Switzerland). To fully induce DC maturation, lipopolysaccharide (LPS) (InvivoGen Corp., USA) was added at 100 ng/mL to the DC cultures on day 6 and cells were incubated for an additional 24 hours. Approximately, 10,000 matured DC and 100,000 purified allogeneic CD4+ T cells (BenTech Bio, USA) were co-cultured with several concentrations of variant PD-L2 IgV-Fc fusion proteins in 96 well round-bottom plates in 200 μL final volume of Ex-Vivo 15 media. Irrelevant human IgG or media only (designated “No Add”) were used as negative controls. As positive controls, either wildtype PDL2-Fc (full PD-L2 extracellular domain), wildtype PD-L2 IgV-Fc and or positive control anti-PD-1 monoclonal antibody (nivolumab) was assessed. On day 5, IFN-gamma secretion in culture supernatants was analyzed using the Human IFN-gamma Duoset ELISA kit (R&D Systems, USA). Optical density was measured on a BioTek Cytation Multimode Microplate Reader (BioTek Corp., USA) and quantitated against titrated rIFN-gamma standard included in the IFN-gamma Duo-set kit (R&D Systems, USA).

Results for the bioactivity studies for exemplary tested variant PD-L2 IgV-Fc are shown in Table 11, which sets forth the calculated levels of IFN-gamma in culture supernatants (pg/mL) at the tested concentration of variant IgV-Fc fusion molecule. Also shown in Table 11 is the fold increase in IFN-gamma levels of each variant IgV-Fc fusion molecule compared to the IFN-gamma levels of the corresponding unmodified (wildtype) IgV-Fc fusion molecule not containing the amino acid substitution(s). The variants are identified with reference to the amino acid substitutions in the IgV of PD-L2 with reference to positions corresponding to positions of the unmodified (wildtype) PD-L2 ECD sequence set forth in SEQ ID NO:31.

Exemplary variant PD-L2 IgV-Fc fusion proteins in Table 11 were further tested in an MLR at various increasing concentrations as indicated in FIG. 9 and FIG. 10. As shown in FIG. 9, exemplary variant PD-L2 IgV-Fc molecules exhibited improved activities to increase IFN-gamma production in an MLR assay. As shown in FIGS. 9 and 10, the exemplary variant PD-L2 IgV-Fc fusion molecules tested show comparable activity to nivolumab in the MLR assay.

TABLE 11 Bioactivity Data of PD-L2 variants selected against PD-1 in MLR. Fold increase IFN over SEQ ID gamma wildtype NO levels PD-L2 PD-L2 mutation(s) (IgV) pg/mL IgV-Fc H15Q 268 1817.1 1.32 N24D 269 1976.3 1.44 E44D 270 1499.4 1.09 V89D 271 1168.1 0.85 Q82R,V89D 272 1617 1.17 E59G,Q82R 273 1511.3 1.1 S39I,V89D 274 1314.5 0.95 S67L,V89D 275 1230.1 0.89 S67L,I85F 276 1281.9 0.93 S67L,I86T 277 1020.4 0.74 H15Q,K65R 278 1510.8 1.1 H15Q,Q72H,V89D 279 1272.2 0.92 H15Q,S67L,R76G 280 1426.2 1.04 H15Q,R76G,185F 281 1725.7 1.25 H15Q,T47A,Q82R 282 1317.9 0.96 H15Q,Q82R,V89D 283 1081.2 0.79 H15Q,C23S,I86T 284 1847.2 1.34 H15Q,S39I,I86T 285 1415.2 1.03 H15Q,R76G,I85F 286 1437.8 1.04 E44D,V89D,W91R 287 1560.1 1.13 I13V,S67L,V89D 288 867.5 0.63 H15Q,S67L,I86T 289 1034.2 0.75 I13V,H15Q,S67L,I86T 290 1014.4 0.74 I13V,H15Q,E44D,V89D 291 1384.2 1.01 I13V,S39I,E44D,Q82R,V89D 292 935.6 0.68 I13V,E44D,Q82R,V89D 293 1009.5 0.73 I13V,Q72H,R76G,I86T 294 1953 1.42 I13V,H15Q,R76G,I85F 295 1528.5 1.11 H15Q,S67L,R76G,I85F 297 1318.7 0.96 H15Q,T47A,Q72H,R76G,I86T 298 1599.6 1.16 H15Q,T47A,Q72H,R76G 299 1462.5 1.06 I13V,H15Q,T47A,Q72H,R76G 300 1469.8 1.07 H15Q,E44D,R76G,185F 301 1391.6 1.01 H15Q,S39I,S67L,V89D 302 1227 0.89 H15Q,N32D,S67L,V89D 303 1285.7 0.93 N32D,S67L,V89D 304 1194 0.87 H15Q,S67L,Q72H,R76G,V89D 305 1061.2 0.77 H15Q,Q72H,Q74R,R76G,I86T 306 933.8 0.68 G28V,Q72H,R76G,I86T 307 1781.6 1.29 I13V,H15Q,S391,E44D,S67L 308 1256.9 0.91 E44D,S67L,Q72H,Q82R,V89D 309 1281.4 0.93 H15Q,V89D 310 1495.4 1.09 H15Q,T47A 311 1637.2 1.19 I13V,H15Q,Q82R 312 1432.9 1.04 I13V,H15Q,V89D 313 1123 0.82 I13V,S67L,Q82R,V89D 314 1372.8 1 I13V,H15Q,Q82R,V89D 315 1596.6 1.16 H15Q,V31M,S67L,Q82R,V89D 316 1206.5 0.88 I13V,H15Q,T47A,Q82R 317 1703.3 1.24 I13V,H15Q,V31A,N45S,Q82R,V89D 318 1723.1 1.25 I13V, T20A, T47A, K65X, Q82R, V89D 319 1378.8 1 H15Q,T47A,H69L,Q82R,V89D 320 1732.5 1.26 I13V,H15Q,T47A,H69L,R76G,V89D 321 1075.5 0.78 I12V,I13V,H15Q,T47A,Q82R,V89D 322 1533.2 1.11 I13V,H15Q,R76G,D77N,Q82R,V89D 323 1187.9 0.86 I13V,H15Q,T47A,R76G,V89D 324 1253.7 0.91 I13V,H15Q,T47A,Q82R,V89D 325 1445.5 1.05 I13V,H15Q,T47A,Q82R,V89D 325 1737 1.26 I13V,H15Q,I36V,T47A,S67L,V89D 327 1357.4 0.99 H15Q,T47A,K65R,S67L,Q82R,V89D 328 1335.3 0.97 H15Q,L33P,T47A,S67L,P71S,V89D 329 1289.1 0.94 I13V,H15Q,Q72H,R76G,I86T 330 1221 0.89 H15Q,T47A,S67L,Q82R,V89D 331 1197.1 0.87 F2L,H15Q,D46E,T47A,Q72H,R76G,Q82R,V89D 332 1170.7 0.85 I13V,H15Q,L33F,T47A,Q82R,V89D 333 1468.4 1.07 I13V,H15Q,T47A,E58G,S67L,Q82R,V89D 334 836.1 0.61 H15Q,N24S,T47A,Q72H,R76G,V89D 335 1091.8 0.79 I13V,H15Q,E44V,T47A,Q82R,V89D 336 1270.5 0.92 H15Q,N18D,T47A,Q72H,V73A,R76G,I86T,V89D 337 1065.8 0.77 I13V,H15Q,T37A,E44D,S48C,S67L,Q82R,V89D 338 1751.7 1.27 H15Q,L33H,S67L,R76G,Q82R,V89D 339 1502 1.09 I13V,H15Q,T47A,Q72H,R76G,I86T 340 1088.1 0.79 H15Q,S39I,E44D,Q72H,V75G,R76G,Q82R,V89D 341 940.9 0.68 H15Q,T47A,S67L,R76G,Q82R,V89D 342 1097.8 0.8 I13V,H15Q,T47A,S67L,Q72H,R76G,Q82R,V89D 343 1559.6 1.13 Wild Type PD-L2 IgV 115 1376.8 1 Full length ECD of PD-L2 31 1173.2 0.85 Full length ECD of PD-L1 30 2190.9 1.59 Nivolumab (anti-PD-1) — 418.9 0.3

Example 8 Additional Affinity Modified IgSF Domains

This examples describe the design, creation, and screening of additional affinity modified CD80 (B7-1), PD-L1, CD155 and CD112 and CD86 (B7-2) immunomodulatory proteins, which are other components of the immune synapse (IS) that have a demonstrated dual role in both immune activation and inhibition. Affinity-modified NKp30 variants also were generated and screened. These examples demonstrate that affinity modification of IgSF domains yields proteins that can act to both increase and decrease immunological activity. Various combinations of those domains fused in pairs (i.e., stacked) with a variant affinity modified PD-L2 to form a Type II immunomodulatory protein to achieve immunomodulatory activity.

Mutant DNA constructs of encoding a variant of the ECD domain of human CD80, or IgV domains of PD-L1, CD155 and CD112 for translation and expression as yeast display libraries were generated substantially as described in Example 1. Target libraries that target specific residues for complete or partial randomization with degenerate codons and/or random libraries were constructed to identify variants of the IgV of CD80 (SEQ ID NO:2039), variants of the ECD or IgV of PD-L1 (SEQ ID NO: 1454), variants of the IgV of CD155 (SEQ ID NO:387), and variants of the IgV of CD112 (SEQ ID NO:795) substantially as described in Example 1. Similar methods also were used to generate libraries of the IgC-like domain of NKp30 (SEQ ID NO:1238).

The degenerate or random library DNA was introduced into yeast substantially as described in Example 2 to generate yeast libraries. The libraries were used to select yeast expressing affinity modified variants of CD80, PD-L1, CD155, CD112, CD86 (B7-2), and NKp30 substantially as described in Example 3. Cells were processed to reduce non-binders and to enrich for CD80, PD-L1, CD155 or CD112, CD86 (B7-2), and NKp30 variants with the ability to bind their exogenous recombinant counter-structure proteins substantially as described in Example 3.

With CD80, CD86 and NKp30 libraries, target ligand proteins were sourced from R&D Systems (USA) as follows: human rCD28.Fc (i.e., recombinant CD28-Fc fusion protein), rPDL1.Fc, rCTLA4.Fc, and rB7H6.Fc. Two-color flow cytometry was performed substantially as described in Example 3. Yeast outputs from the flow cytometric sorts were assayed for higher specific binding affinity. Sort output yeast were expanded and re-induced to express the particular IgSF affinity modified domain variants they encode. This population then can be compared to the parental, wild-type yeast strain, or any other selected outputs, such as the bead output yeast population, by flow cytometry.

In the case of NKp30 yeast variants selected for binding to B7-H6, the F2 sort outputs gave MFI values of 533 when stained with 16.6 nM rB7H6.Fc, whereas the parental NKp30 strain MFI was measured at 90 when stained with the same concentration of rB7H6.Fc (6-fold improvement).

Among the NKp30 variants that were identified, was a variant that contained mutations L30V/A60V/S64P/S86G with reference to positions in the NKp30 extracellular domain corresponding to positions set forth in SEQ ID NO:54.

For CD80 variants provided in Table 12A-B, CD80 libraries consisted of positive selection with the desired counter structure CTLA4 and negative selection with the counter structure CD28.

For CD155 variants provided in Table 13A, CD155 libraries were selected against each of TIGIT, CD96, and CD226, separately. For CD155 variants provided in Table 13B-F, selection involved two positive selections with the desired counter structures TIGIT and CD96 followed by one negative selection with the counter structure CD226 to select away from CD226 and improve binding specificity of the variant CD155. Selection was performed essentially as described in Example 3 above except the concentrations of the counter structures (TIGIT/CD96) and selection stringency of the positive sorts were varied to optimize lead identification. The concentration of CD226 for the negative selection was kept at 100 nM.

For CD112 variants provided in Table 14A, CD112 libraries were selected against each of TIGIT, CD112R, and CD226, separately. For additional CD112 variants provided in Table 14B-14C, selection involved two positive selections with the desired counter structures TIGIT and CD112R followed by one negative selection with the counter structure CD226 to select away from CD226 and improve binding specificity of the variant CD112. Selection was performed essentially as described in Example 3 above except the concentrations of the counter structures (TIGIT/CD112R) and selection stringency of the positive sorts were varied to optimize lead identification. The concentration of CD226 for the negative selection was kept at 100 nM.

For PD-L1 variants provided in Table 15, yeast display targeted or random PD-L1 libraries were selected against PD-1. This was then followed by two to three rounds of flow cytometry sorting using exogenous counter-structure protein staining to enrich the fraction of yeast cells that displays improved binders. Alternatively, for PD-L1, selections were performed with human rCD80.Fc (i.e., human recombinant CD80 Fc fusion protein from R&D Systems, USA). Selections were carried out largely as described for PD-1. Magnetic bead enrichment and selections by flow cytometry are essentially as described in Miller K. D., Current Protocols in Cytometry 4.7.1-4.7.30, July 2008. PD-L1 variants in Table 15A-B were assessed for binding to cell-expressed counter structures. Additional PD-L1 variants identified in the screen as described above are set forth in Table 15C.

Exemplary selection outputs were reformatted as immunomodulatory proteins containing an affinity modified (variant) IgV of CD80, variant IgV of PD-L1, variant IgV of CD155, variant IgV of CD112, each fused to an Fc molecule (variant ECD-Fc fusion molecules or variant IgV-Fc fusion molecules) substantially as described in Example 4 and the Fc-fusion protein was expressed and purified substantially as described in Example 5.

Binding of exemplary IgSF domain variants to cell-expressed counter structures was then assessed substantially as described in Example 6. Cells expressing cognate binding partners were produced and binding studies and flow cytometry were carried out substantially as described in Example 6. In addition, the bioactivity of the Fc-fusion variant protein was characterized by either mixed lymphocyte reaction (MLR) or anti-CD3 coimmobilization assay substantially as described in Example 6.

As above, for each Table, the exemplary amino acid substitutions are designated by amino acid position number corresponding to the respective reference unmodified ECD sequence (Table 2). The amino acid position is indicated in the middle, with the corresponding unmodified (e.g. wild-type) amino acid listed before the number and the identified variant amino acid substitution listed (or inserted designated by a) after the number.

Also shown is the binding activity as measured by the Mean Fluorescence Intensity (MFI) value for binding of each variant Fc-fusion molecule to cells engineered to express the cognate counter structure ligand and the ratio of the MFI compared to the binding of the corresponding unmodified Fc fusion molecule not containing the amino acid substitution(s) to the same cell-expressed counter structure ligand. The functional activity of the variant Fc-fusion molecules to modulate the activity of T cells also is shown based on the calculated levels of IFN-gamma in culture supernatants (pg/mL) generated either i) with the indicated variant Fc fusion molecule coimmoblized with anti-CD3 or ii) with the indicated variant Fc fusion molecule in an MLR assay. The Tables also depict the ratio of IFN-gamma produced by each variant ECD-Fc or IgV-Fc compared to the corresponding unmodified ECD-Fc or IgV-Fc in the functional assays.

As shown in Tables 12A-15, the selections resulted in the identification of a number of PD-L1, CD155, CD112, and CD80 IgSF domain variants that were affinity-modified to exhibit increased binding for at least one, and in some cases more than one, cognate counter structure ligand. In addition, the results showed that affinity modification of the variant molecules also exhibited improved activities to both increase and decrease immunological activity depending on the format of the molecule.

TABLE 12A Variant CD80 Binding to HEK293 Cells Transfected with CTLA4, CD28 or PD-L1 CTLA4 CD28 PD-L1 SEQ ID MFI at Fold MFI Fold MFI at Fold NO 66.6 change at 66.6 change 22.2 change Ratio of CD80 mutation(s) (IgV) nM to WT nM to WT nM to WT CTLA4:CD28 L70P 1114 Not tested I30F/L70P 1115 Not tested Q27H/T41S/A71D 1116 368176 2.3 25051 1.01 24181 N/A 14.7 I30T/L70R 1117 2234 0.0 2596 0.10 5163 N/A 0.9 T13R/C16R/L70Q/A71D 1118 197357 1.2 16082 0.65 9516 N/A 12.3 T57I 1119 393810 2.4 23569 0.95 3375 N/A 16.7 M43I/C82R 1120 3638 0.0 3078 0.12 7405 N/A 1.2 V22L/M38V/M47T/A71D/ 1121 175235 1.1 3027 0.12 6144 N/A 57.9 L85M I30V/T57I/L70P/A71D/ 1122 116085 0.7 10129 0.41 5886 N/A 11.5 A91T V22I/L70M/A71D 1123 163825 1.0 22843 0.92 33404 N/A 7.2 N55D/L70P/E77G 1124 Not tested T57A/I69T 1125 Not tested N55D/K86M 1126 3539 0.0 3119 0.13 5091 N/A 1.1 L72P/T79I 1127 50176 0.3 3397 0.14 6023 N/A 14.8 L70P/F92S 1128 4035 0.0 2948 0.12 6173 N/A 1.4 T79P 1129 2005 0.0 2665 0.11 4412 N/A 0.8 E35D/M47I/L65P/D90N 1130 4411 0.0 2526 0.10 4034 N/A 1.7 L25S/E35D/M47I/D90N 1131 61265 0.4 4845 0.20 20902 N/A 12.6 Q27X*/S44P/I67T/P74S/ 1132 195637 1.2 17524 0.71 17509 N/A 11.2 E81G/E95D A71D 1133 220090 1.4 16785 0.68 29642 N/A 13.1 T13A/Q27X*/I61N/A71D 1134 195061 1.2 17519 0.71 21717 N/A 11.1 E81K/A91S 1135 98467 0.6 3309 0.13 44557 N/A 29.8 A12V/M47V/L70M 1136 81616 0.5 7400 0.30 31077 N/A 11.0 K34E/T41A/L72V 1137 88982 0.6 3755 0.15 35293 N/A 23.7 T41S/A71D/V84A 1138 103010 0.6 5573 0.22 83541 N/A 18.5 E35D/A71D 1139 106069 0.7 18206 0.73 40151 N/A 5.8 E35D/M47I 1140 353590 2.2 14350 0.58 149916 N/A 24.6 K36R/G78A 1141 11937 0.1 2611 0.11 5715 N/A 4.6 Q33E/T41A 1142 8292 0.1 2442 0.10 3958 N/A 3.4 M47V/N48H 1143 207012 1.3 14623 0.59 145529 N/A 14.2 M47L/V68A 1144 74238 0.5 13259 0.53 11223 N/A 5.6 S44P/A71D 1145 8839 0.1 2744 0.11 6309 N/A 3.2 Q27H/M43I/A71D/R73S 1146 136251 0.8 12391 0.50 8242 N/A 11.0 E35D/T57I/L70Q/A71D 1148 121901 0.8 21284 0.86 2419 N/A 5.7 M47I/E88D 1149 105192 0.7 7337 0.30 97695 N/A 14.3 M42I/I61V/A71D 1150 54478 0.3 6074 0.24 4226 N/A 9.0 P51A/A71D 1151 67256 0.4 4262 0.17 5532 N/A 15.8 H18Y/M47I/T57I/A71G 1152 136455 0.8 20081 0.81 13749 N/A 6.8 V20I/M47V/T57I/V84I 1153 183516 1.1 26922 1.08 3583 N/A 6.8 WT 2039 161423 1.0 24836 1.00 Not N/A 6.5 tested *Stop codon at indicated position

TABLE 12B Variant CD80 Binding to HEK293 Cells Transfected with CTLA4, CD28 or PD-L1 CTLA4 CD28 PD-L1 SEQ MFI at Fold MFI at Fold MFI at Fold Ratio of ID NO 66.6 change 66.6 change 22.2 change CTLA4: CD80 mutation(s) (IgV) nM to WT nM to WT nM to WT CD28 V20I/M47V/A71D 1154 149937 7.23 15090 9.33 9710 5.48 9.9 A71D/L72V/E95K 1155 140306 6.77 6314 3.90 8417 4.75 22.2 V22L/E35G/A71D/L72P 1156 152588 7.36 8150 5.04 1403 0.79 18.7 E35D/A71D 1157 150330 7.25 14982 9.26 13781 7.77 10.0 E35D/I67L/A71D 1158 146087 7.04 11175 6.91 9354 5.28 13.1 T13R/M42V/M47I/A71D 1160 108900 5.25 16713 10.33 1869 1.05 6.5 E35D 1161 116494 5.62 3453 2.13 25492 14.38 33.7 E35D/M47I/L70M 1162 116531 5.62 14395 8.90 49131 27.71 8.1 E35D/A71/L72V 1163 134252 6.47 11634 7.19 13125 7.40 11.5 E35D/M43L/L70M 1164 102499 4.94 3112 1.92 40632 22.92 32.9 A26P/E35D/M43I/L85Q/E88D 1165 83139 4.01 5406 3.34 9506 5.36 15.4 E35D/D46V/L85Q 1166 85989 4.15 7510 4.64 38133 21.51 11.4 Q27L/E35D/M47I/T57I/L70Q/E88D 1167 59793 2.88 14011 8.66 1050 0.59 4.3 Q27H/E35G/A71D/L72P/T79I 1159 85117 4.10 10317 6.38 1452 0.82 8.3 M47V/I69F/A71D/V83I 1168 76944 3.71 15906 9.8.3 3399 1.92 4.8 E35D/T57A/A71D/L85Q 1169 85724 4.13 3383 2.09 1764 0.99 25.3 H18Y/A26T/E35D/A71D/L85Q 1170 70878 3.42 6487 4.01 8026 4.53 10.9 E35D/M47L 1171 82410 3.97 11508 7.11 58645 33.08 7.2 E23D/M42V/M43I/I58V/L70R 1172 37331 1.80 10910 6.74 2251 1.27 3.4 V68M/L70M/A71D/E95K 1173 56479 2.72 10541 6.51 38182 21.53 5.4 N55I/T57I/I69F 1174 2855 0.14 1901 1.17 14759 8.32 1.5 E35D/M43I/A71D 1175 63789 3.08 6369 3.94 27290 15.39 10.0 T41S/T57I/L70R 1176 59844 2.89 4902 3.03 19527 11.01 12.2 H18Y/A71D/L72P/E88V 1177 68391 3.30 8862 5.48 1085 0.61 7.7 V20I/A71D 1178 60323 2.91 10500 6.49 3551 2.00 5.7 E23G/A26S/E35D/T62N/A71D/L72V/L85M 1179 59025 2.85 5484 3.39 10662 6.01 10.8 A12T/E24D/E35D/D46V/I61V/L72P/E95V 1180 63738 3.07 7411 4.58 1221 0.69 8.6 V22L/E35D/M43L/A71G/D76H 1181 2970 0.14 1498 0.93 1851 1.04 2.0 E35G/K54E/A71D/L72P 1182 71899 3.47 3697 2.29 1575 0.89 19.4 L70Q/A71D 1183 45012 2.17 18615 11.50 1692 0.95 2.4 A26E/E35D/M47L/L85Q 1184 40325 1.94 2266 1.40 55548 31.33 17.8 D46E/A71D 1185 69674 3.36 16770 10.36 22777 12.85 4.2 Y31H/E35D/T41S/V68L/K93R/R94W 1186 3379 0.16 2446 1.51 18863 10.64 1.4 WT CD80 IgV Fc 2039 20739 1.00 1618 1.00 1773 1.00 12.8 WT CD80 ECD Fc — 72506 3.50 3072 1.90 4418 2.49 23.6

TABLE 13A Variant CD155 selected against cognate binding partners. Molecule sequences, binding data, and costimulatory bioactivity data. Anti-CD3 IFN- TIGIT Mock gamma CD226 tfxn CD96 Expi293 (pg/mL) tfxn MFI MFI MFI (Anti-CD3 SEQ MFI (TIGIT (CD96 (Mock IFN- ID (CD226 MFI MFI MFI gamma CD155 NO MFI parental parental parental parental mutations (IgV) parental ratio) ratio) ratio) ratio) P18S, P64S, 388 497825 247219 140065 3528 270.1 F91S (133.7) (91.1) (45.4) (1.2) (0.7) P18S, F91S, 389 26210 75176 10867 2130 364.2 L104P (7.0) (27.7) (3.5) (0.7) (0.9) L44P 390 581289 261931 152252 3414 277.6 (156.1) (96.5) (49.4) (1.2) (0.7) A56V 391 455297 280265 161162 2601 548.2 (122.3) (103.2) (52.2) (0.9) (1.4) P18L, L79V, 392 5135 4073 3279 2719 1241.5 F91S (1.4) (1.5) (1.1) (0.9) (3.2) P18S, F91S 393 408623 284190 147463 3348 760.6 (109.8) (104.7) (47.8) (1.1) (2.0) P18T, F91S 394 401283 223985 157644 3065 814.7 (107.8) (82.5) (51.1) (1.1) (2.1) P18T, S42P, 395 554105 223887 135395 3796 539.7 F91S (148.8) (82.5) (43.9) (1.3) (1.4) G7E, P18T, 396 12903 12984 7906 2671 275.9 Y30C, F91S (3.5) (4.8) (2.6) (0.9) (0.7) P18T, F91S, 397 438327 287315 167583 4012 307.2 G111D (117.7) (105.8) (54.3) (1.4) (0.8) P18S, F91P 398 4154 3220 2678 2816 365.7 (1.1) (1.2) (0.9) (1.0) (0.9) P18T, F91S, 399 394546 298680 193122 2926 775.4 F108L (106.0) (110.0) (62.6) (1.0) (2.0) P18T, T45A, 400 435847 222044 191026 2948 1546.8 F91S (117.1) (81.8) (61.9) (1.0) (4.0) P18T, F91S, 401 3589 2942 2509 2390 1273.2 R94H (1.0) (1.1) (0.8) (0.8) (3.3) P18S, Y30C, 402 382352 276358 56934 3540 426.5 F91S (102.7) (101.8) (18.5) (1.2) (1.1) A81V, L83P 403 4169 2912 2616 2993 339.7 (1.1) (1.1) (0.8) (1.0) (0.9) L88P 404 65120 74845 35280 2140 969.2 (17.5) (27.6) (11.4) (0.7) (2.5) Wild type 387 3723 2715 3085 2913 389.6 (1.0) (1.0) (1.0) (1.0) (1.0) R94H 405 18905 104013 11727 1663 372.6 (5.1) (38.3) (3.8) (0.6) (1.0) A13E, P18S, 406 357808 179060 118570 2844 349.2 A56V, F91S (96.1) (66.0) (38.4) (1.0) (0.9) P18T, F91S, 407 38487 46313 22718 2070 1574.5 V115A (10.3) (17.1) (7.4) (0.7) (4.0) P18T, Q60K 408 238266 173730 154448 4778 427.2 (64.0) (64.0) (50.1) (1.6) (1.1)

TABLE 13B Additional CD155 Variants and Binding Data. TIGIT CD226 CD112R CD96 SEQ Fold Fold Fold Fold ID ↑ to ↑ to ↑ to ↑ to CD155 NO MFI at WT MFI at WT MFI at WT MFI at WT Mutation(s) (IgV) 100 nM ECD 100 nM ECD 100 nM ECD 100 nM ECD S52M 603 1865.3 0.00 1901.0 0.01 1553.4 0.87 1609.8 0.02 T45Q, S52L, 604 2287.0 0.01 2390.4 0.01 1735.1 0.97 1575.1 0.02 L104E, G111R S42G 605 4837.5 0.01 2448.1 0.01 1815.4 1.02 1699.6 0.02 Q62F 606 2209.5 0.01 2572.1 0.01 2706.5 1.52 2760.7 0.03 S52Q 607 2288.1 0.01 2022.3 0.01 1790.1 1.00 1822.3 0.02 S42A, L104Q, 608 1923.7 0.00 1901.7 0.01 1815.1 1.02 1703.8 0.02 G111R S42A, S52Q, 609 1807.5 0.00 2157.2 0.01 1894.4 1.06 1644.0 0.02 L104Q, G111R S52W, L104E 610 1938.2 0.00 1905.6 0.01 2070.6 1.16 1629.5 0.02 S42C 611 1914.0 0.00 2096.1 0.01 1685.0 0.95 1592.4 0.02 S52W 612 1991.6 0.00 2037.3 0.01 1612.8 0.90 1712.9 0.02 S52M, L104Q 613 2666.6 0.01 2252.2 0.01 1706.0 0.96 1633.1 0.02 S42L, S52L, 614 2021.4 0.00 2643.8 0.02 1730.1 0.97 2318.7 0.02 Q62F, L104Q S42W 615 2434.5 0.01 2133.4 0.01 2325.7 1.30 2555.4 0.03 S42Q 616 2073.5 0.00 2225.9 0.01 1905.1 1.07 2143.1 0.02 S52L 617 2224.8 0.01 2676.3 0.02 2038.6 1.14 2043.2 0.02 S52R 618 4395.4 0.01 3964.4 0.02 2741.7 1.54 4846.9 0.05 L104E 619 3135.4 0.01 2264.2 0.01 1803.5 1.01 1556.7 0.02 G111R 620 2082.7 0.00 2791.3 0.02 2470.9 1.39 3317.1 0.03 S52E 621 2655.4 0.01 2599.8 0.02 1904.9 1.07 1799.0 0.02 Q62Y 622 2528.6 0.01 2621.4 0.02 1918.4 1.08 1827.5 0.02 T45Q, S52M, 623 79498.2 0.19 143238.5 0.83 2600.6 1.46 6310.4 0.06 L104E S42N, L104Q, 624 2432.1 0.01 2311.3 0.01 1847.4 1.04 1958.3 0.02 G111R S52M, V57L 625 1760.7 0.00 2431.6 0.01 2006.9 1.13 1858.7 0.02 S42N, S52Q, 626 2402.7 0.01 2152.0 0.01 1855.0 1.04 1737.6 0.02 Q62F S42A, S52L, 627 2262.7 0.01 1889.4 0.01 1783.2 1.00 1606.2 0.02 L104E, G111R S42W, S52Q, 628 1961.4 0.00 2138.3 0.01 1844.9 1.03 1699.6 0.02 V57L, Q62Y L104Q 629 10314.4 0.02 3791.4 0.02 2119.9 1.19 1542.6 0.02 S42L, S52Q, 630 1946.9 0.00 6474.3 0.04 1749.0 0.98 1702.2 0.02 L104E S42C, S52L 631 1762.5 0.00 2147.3 0.01 1663.4 0.93 1484.7 0.01 S42W, S52R, 632 1918.8 0.00 2300.1 0.01 1824.6 1.02 1756.0 0.02 Q62Y, L104Q T45Q, S52R, 633 121636.9 0.29 142381.2 0.82 2617.9 1.47 3748.2 0.04 L104E S52R, Q62F, 634 2969.2 0.01 3171.6 0.02 1725.4 0.97 2362.3 0.02 L104Q, G111R T45Q, S52L, 635 2857.7 0.01 5943.5 0.03 1496.8 0.84 1533.3 0.02 V57L, L104E S52M, Q62Y 636 1926.6 0.00 2000.3 0.01 1771.6 0.99 1651.1 0.02 Q62F, L104E, 637 1966.4 0.00 2043.5 0.01 1701.9 0.95 1524.8 0.02 G111R T45Q, S52Q 638 4812.8 0.01 5787.5 0.03 1765.6 0.99 2451.3 0.02 S52L, L104E 639 4317.8 0.01 2213.9 0.01 1756.9 0.99 1829.3 0.02 S42V, S52E 640 2055.0 0.00 2272.6 0.01 1808.0 1.01 2530.2 0.03 T45Q, S52R, 641 4092.3 0.01 2075.2 0.01 1793.6 1.01 2336.6 0.02 G111R S42G, S52Q, 642 2010.1 0.00 2019.2 0.01 1706.4 0.96 1707.6 0.02 L104E, G111R S42N, S52E, 643 1784.2 0.00 1743.6 0.01 1690.1 0.95 1538.7 0.02 V57L, L104E Wildtype 387 1964.7 0.00 2317.1 0.01 2169.6 1.22 1893.4 0.02 S42C, S52M, 644 1861.0 0.00 2084.2 0.01 1592.3 0.89 1481.3 0.01 Q62F S42L 645 1930.4 0.00 2187.2 0.01 1743.2 0.98 1618.4 0.02 Wildtype 387 2182.6 0.01 2374.5 0.01 1743.1 0.98 1680.4 0.02 S42A 646 1929.2 0.00 2188.6 0.01 1733.7 0.97 1623.6 0.02 S42G, S52L, 647 1924.3 0.00 2157.6 0.01 1661.3 0.93 1642.1 0.02 Q62F, L104Q S42N 648 1817.4 0.00 1910.9 0.01 1699.7 0.95 1691.5 0.02 CD155 IgV Fc 387 4690 0.01 4690 0.03 2941 1.65 3272 0.03 Wildtype 47 423797 1.00 172839 1.00 1783 1.00 99037 1.00 CD155 (ECD) ECD-Fc Anti-human — 1506.3 0.00 3774 0.02 1587 0.89 1618 0.02 Fc PE

TABLE 13C Additional C155 Variants and Binding Data. TIGIT CD226 CD96 SEQ MFI Fold MFI Fold MFI Fold ID at Increase at Increase at Increase NO 100 to WT 100 to WT 100 to WT CD155 Mutation(s) (IgV) nM ECD nM ECD nM ECD P18T, S65A, S67V, F91S 649 297843 1.99 351195 3.22 128180 1.68 P18F, T39A, T45Q, T61R, 650 Little to no protein produced S65N, S67L, E73G, R78G P18T, T45Q, T61R, S65N, 651 224682 1.50 270175 2.48 22820 0.30 S67L P18F, S65A, S67V, F91S 652 534106 3.57 350410 3.21 144069 1.89 P18F, T45Q, T61R, S65N, 653 Little to no protein produced S67L, F91S, L104P P18S, L79P, L104M 654 342549 2.29 320823 2.94 107532 1.41 P18S, L104M 655 449066 3.00 295126 2.70 121266 1.59 L79P, L104M 656 3210 0.02 8323 0.08 2894 0.04 P18T, T45Q, L79P 657 542878 3.63 371498 3.40 193719 2.55 P18T, T45Q, T61R, S65H, 658 312337 2.09 225439 2.07 152903 2.01 S67H P18T, A81E 659 Little to no protein produced P18S, D23Y, E37P, S52G, 660 Little to no protein produced Q62M, G80S, A81P, G99Y, S112N A13R, D23Y, E37P, S42P, 661 4161 0.03 11673 0.11 5762 0.08 Q62Y, A81E A13R, D23Y, E37P, G99Y, 662 Little to no protein produced S112N A13R, D23Y, E37P, Q62M, 663 Little to no protein produced A77V, G80S, A81P, G99Y P18L, E37S, Q62M, G80S, 664 5900 0.04 14642 0.13 3345 0.04 A81P, G99Y, S112N P18S, L104T 665 321741 2.15 367470 3.37 108569 1.43 P18S, Q62H, L79Q, F91S 666 283357 1.89 324877 2.98 125541 1.65 P18S, F91S 393 222780 1.49 300049 2.75 48542 0.64 T45Q, S52K, Q62F, L104Q, 667 Little to no protein produced G111R T45Q, S52Q, Q62Y, L104Q, 668 Little to no protein produced G111R T45Q, S52Q, Q62Y, L104E, 669 Little to no protein produced G111R V57A, T61M, S65W, S67A, 670 Little to no protein produced E96D, L104T P18L, V57T, T61S, S65Y, 671 278178 1.86 276870 2.54 121499 1.60 S67A, L104T P18T, T45Q 672 326769 2.18 357515 3.28 92389 1.21 P18L, V57A, T61M, S65W, 673 Little to no protein produced S67A, L104T T61M, S65W, S67A, L104T 674 360915 2.41 417897 3.83 148954 1.96 P18S, V41A, S42G, T45G, 675 3821 0.03 11449 0.10 3087 0.04 L104N P18H, S42G, T45I, S52T, 676 5066 0.03 177351 1.63 3700 0.05 G53R, S54H, V57L, H59E, T61S, S65D, E68G, L104N P18S, S42G, T45V, F58L, 677 14137 0.09 15175 0.14 15324 0.20 S67W, L104N P18S, T45I, L104N 678 141745 0.95 298011 2.73 97246 1.28 P18S, S42G, T45G, L104N, 679 29387 0.20 117965 1.08 15884 0.21 V106A P18H, H40R, S42G, T45I, 680 12335 0.08 14657 0.13 15779 0.21 S52T, G53R, S54H, V57L, H59E, T61S, S65D, E68G, L104Y, V106L, F108H E37V, S42G, T45G, L104N 681 Little to no protein produced P18S, T45Q, L79P, L104T 682 206674 1.38 285512 2.62 87790 1.15 P18L, Q62R 683 66939 0.45 25063 0.23 10928 0.14 A13R, D23Y, E37P, S42L, 684 Little to no protein produced S52G, Q62Y, A81E P18L, H49R, L104T, D116N 685 167980 1.12 214677 1.97 62451 0.82 A13R, D23Y, E37P, Q62M, 686 Little to no protein produced G80S, A81P, L104T S65T, L104T 687 205942 1.38 187147 1.71 65207 0.86 A13R, D23Y, E37P, S52G, 688 Little to no protein produced V57A, Q62M, K70E, L104T P18L, A47V, Q62Y, E73D, 689 146142 0.98 248926 2.28 73956 0.97 L104T H40T, V41M, A47V, S52Q, 690 Little to no protein produced Q62L, S65T, E73R, D97G, E98S, L104T, D116N P18L, S42P, T45Q, T61G, 691 153536 1.03 402503 3.69 53044 0.70 S65H, S67E, L104T, D116N P18S, H40T, V41M, A47V, 692 Little to no protein produced S52Q, Q62L, S65T, E73R, L104M, V106A H40T, V41M, A47V, S52Q, 693 Little to no protein produced Q62L, S65T, E68G, E73R, D97G, E98S, L104T T45Q, S52E, L104E 694 Little to no protein produced T45Q, S52E, Q62F, L104E 695 132850 0.89 276434 2.53 14558 0.19 Wildtype CD155 ECD-Fc 47 149692 1.00 109137 1.00 76083 1.00 (ECD) Anti-human Fc PE — 2287 0.02 4799 0.04 2061 0.03

TABLE 13D Additional CD155 Variants and Binding Data. TIGIT CD226 CD96 SEQ MFI Fold Fold MFI Fold ID at Increase MFI at Increase at Increase NO 100 to WT 100 to WT 100 to WT CD155 Mutations (IgV) nM IgV nM IgV nM IgV P18F, T26M, L44V, 696 117327 1.2 1613 0.1 1629 0.1 Q62K, L79P, F91S, L104M, G111D P18S, T45S, T61K, 697 124936 1.3 2114 0.1 2223 0.1 S65W, S67A, F91S, G111R P18S, L79P, 698 110512 1.1 18337 0.9 22793 1.3 L104M, T107M P18S, S65W, S67A, 696 101726 1.0 1605 0.1 2571 0.1 M90V, V95A, L104Q, G111R Wildtype CD155- 47 98935 1.0 20029 1.0 17410 1.0 ECD (ECD)

TABLE 13E Additional CD155 Variants and Binding Data. TIGIT CD226 CD96 Fold Fold Fold SEQ Change Change Change ID MFI at from MFI at from MFI at from NO 11.1 CD155- 11.1 CD155- 11.1 CD155- CD155 Mutations (IgV) nM ECD nM ECD nM ECD P18S, A47G, L79P, F91S, 1550 56,409 1.19 1,191 0.08 25,362 1.49 L104M, T107A, R113W P18T, D23G, S24A, N35D, 1551 128,536 2.72 987 0.06 3,497 0.20 H49L, L79P, F91S, L104M, G111R V9L, P18S, Q60R, V75L, 1552 125,329 2.65 986 0.06 959 0.06 L79P, R89K, F91S, L104E, G111R P18S, H49R, E73D, L79P, 1553 Little to no protein produced N85D, F91S, V95A, L104M, G111R V11A, P18S, L79P, F91S, 1554 48,246 1.02 974 0.06 923 0.05 L104M, G111R V11A, P18S, S54R, Q60P, 1555 190,392 4.02 1,019 0.07 1,129 0.07 Q62K, L79P, N85D, F91S, T107M P18T, S52P, S65A, S67V, 1556 121,611 2.57 986 0.06 16,507 0.97 L79P, F91S, L104M, G111R P18T, M36T, L79P, F91S, 1557 150,015 3.17 1,029 0.07 2,514 0.15 G111R D8G, P18S, M36I, V38A, 1558 79,333 1.68 1,026 0.07 2,313 0.14 H49Q, A76E, F91S, L104M, T107A, R113W P18S, S52P, S65A, S67V, 1559 23,766 0.50 1,004 0.07 1,080 0.06 L79P, F91S, L104M, T107S, R113W T15I, P18T, L79P, F91S, 1560 55,498 1.17 1,516 0.10 1,030 0.06 L104M, G111R P18F, T26M, L44V, Q62K, 1561 213,640 4.51 991 0.06 1,276 0.07 L79P, E82D, F91S, L104M, G111D P18T, E37G, G53R, Q62K, 1562 251,288 5.31 2,001 0.13 45,878 2.69 L79P, F91S, E98D, L104M, T107M P18L, K70E, L79P, F91S, 1563 62,608 1.32 1,117 0.07 973 0.06 V95A, G111R V9I, Q12K, P18F, S65A, 1564 81,932 1.73 803 0.05 68,295 4.00 S67V, L79P, L104T, G111R, S112I P18F, S65A, S67V, F91S, 1565 30,661 0.65 901 0.06 3,193 0.19 L104M, G111R V9I, V10I, P18S, F20S, 1566 151,489 3.20 973 0.06 974 0.06 T45A, L79P, F91S, L104M, F108Y, G111R, S112V V9L, P18L, L79P, M90I, 1567 155,279 3.28 910 0.06 10,568 0.62 F91S, T102S, L104M, G111R P18C, T26M, L44V, M55I, 1568 137,521 2.91 973 0.06 111,085 6.51 Q62K, L79P, F91S, L104M, T107M V9I, P18T, D23G, L79P, 1569 151,426 3.20 897 0.06 2,725 0.16 F91S, G111R P18F, L79P, M90L, F91S, 1570 125,639 2.66 917 0.06 3,939 0.23 V95A, L104M, G111R P18F, L79P, M90L, F91S, 1570 115,156 2.43 1,073 0.07 2,464 0.14 V95A, L104M, G111R P18T, M36T, S65A, S67E, 1571 10,616 0.22 1,130 0.07 963 0.06 L79Q, A81T, F91S, G111R V9L, P18T, Q62R, L79P, 1572 195,111 4.12 835 0.05 1,497 0.09 F91S, L104M, G111R CD155-ECD-Fc 47 47,319 1.00 15,421 1.00 17,067 1.00 (ECD) Fc Control 1189 2,298 0.05 1,133 0.07 996 0.06

TABLE 13F Additional CD155 Variants and Binding Data. TIGIT CD226 CD112R CD96 Fold Fold Fold Fold SEQ Change Change Change Change ID from from from from NO MFI at CD155- MFI at CD155- MFI at CD155- MFI at CD155- CD155 Mutations (IgV) 25 nM ECD 25 nM ECD 25 nM ECD 25 nM ECD P18T, G19D, M36T, S54N, 1691 905 0.02 748 0.02 1276 1.56 726 0.01 L79P, L83Q, F91S, T107M, F108Y V9L, P18L, M55V, S69L, 1692 58656 1.34 11166 0.29 920 1.13 67364 1.39 L79P, A81E, F91S, T107M P18F, H40Q, T61K, Q62K, 1693 108441 2.48 853 0.02 918 1.13 8035 0.17 L79P, F91S, L104M, T107V P18S, Q32R, Q62K, R78G, 1694 5772 0.13 701 0.02 843 1.03 831 0.02 L79P, F91S, T107A, R113W Q12H, P18T, L21S, G22S, 1695 1084 0.02 687 0.02 876 1.07 818 0.02 V57A, Q62R, L79P, F91S, T107M V9I, P18S, S24P, H49Q, 1696 69926 1.60 1089 0.03 1026 1.26 43856 0.90 F58Y, Q60R, Q62K, L79P, F91S, T107M P18T, W46C, H49R, S65A, 1697 918 0.02 640 0.02 803 0.98 717 0.01 S67V, A76T, L79P, S87T, L104M P18S, S42T, E51G, L79P, 1698 12630 0.29 707 0.02 857 1.05 1050 0.02 F91S, G92W, T107M P18S, S42T, E51G, L79P, 1698 7476 0.17 851 0.02 935 1.15 924 0.02 F91S, G92W, T107M V10F, T15S, P18L, R48Q, 1699 1168 0.03 792 0.02 901 1.10 998 0.02 L79P, F91S, T107M, V115M P18S, L21M, Y30F, N35D, 1700 1377 0.03 743 0.02 946 1.16 1033 0.02 R84W, F91S, T107M, D116G P18F, E51V, S54G, Q60R, 1701 46090 1.05 15701 0.41 1012 1.24 61814 1.27 L79Q, E82G, S87T, M90I, F91S, G92R, T107M Q16H, P18F, F91S, T107M 1702 Little to no protein produced P18T, D23G, Q60R, S67L, 1703 64091 1.47 30931 0.81 874 1.07 108875 2.24 L79P, F91S, T107M, V115A D8G, V9I, V11A, P18T, 1704 52508 1.20 9483 0.25 817 1.00 97770 2.01 T26M, S52P, L79P, F91S, G92A, T107L, V115A V9I, P18F, A47E, G50S, 1705 55167 1.26 54341 1.43 752 0.92 102115 2.10 E68G, L79P, F91S, T107M P18S, M55I, Q62K, S69P, 1706 Little to no protein produced L79P, F91S, T107M P18T, T39S, S52P, S54R, 1707 45927 1.05 744 0.02 1038 1.27 1225 0.03 L79P, F91S, T107M P18S, D23N, L79P, F91S, 1708 Little to no protein produced T107M, S114N P18S, P34S, E51V, L79P, 1709 7917 0.18 769 0.02 853 1.04 892 0.02 F91S, G111R P18S, H59N, V75A, L79P, 1710 800 0.02 676 0.02 915 1.12 759 0.02 A81T, F91S, L104M, T107M P18S, W46R, E68D, L79P, 1711 1359 0.03 717 0.02 798 0.98 737 0.02 F91S, T107M, R113G V9L, P18F, T45A, S65A, 1712 130274 2.98 153569 4.04 812 1.00 85605 1.76 S67V, R78K, L79V, F91S, T107M, S114T P18T, M55L, T61R, L79P, 1713 133399 3.05 1906 0.05 827 1.01 57927 1.19 F91S, V106I, T107M T15I, P18S, V33M, N35F, 1714 7550 0.17 1015 0.03 789 0.97 2709 0.06 T39S, M55L, R78S, L79P, F91S, T107M P18S, Q62K, K70E, L79P, 1715 1951 11173 0.26 691 0.02 735 0.90 0.04 F91S, G92E, R113W P18F, F20I, T26M, A47V, 1716 136088 3.11 54026 1.42 1401 1.72 96629 1.99 E51K, L79P, F91S P18T, D23A, Q60H, L79P, 1717 43795 1.00 98241 2.58 888 1.09 70891 1.46 M90V, F91S, T107M P18S, D23G, C29R, N35D, 1718 1599 0.04 1030 0.03 1115 1.37 1944 0.04 E37G, M55I, Q62K, S65A, S67G, R78G, L79P, F91S, L104M, T107M, Q110R A13E, P18S, M36R, Q62K, 1719 Little to no protein produced S67T, L79P, N85D, F91S, T107M V9I, P18T, H49R, L79P, 1720 46375 1.06 76851 2.02 794 0.97 80210 1.65 N85D, F91S, L104T, T107M V9A, P18F, T61S, Q62L, 1721 26109 0.60 891 0.02 825 1.01 2633 0.05 L79P, F91S, G111R D8E, P18T, T61A, L79P, 1722 Little to no protein produced F91S, T107M P18S, V41A, H49R, S54C, 1723 1098 0.03 830 0.02 876 1.07 1678 0.03 L79S, N85Y, L88P, F91S, L104M, T107M V11E, P18H, F20Y, V25E, 1724 979 0.02 846 0.02 844 1.03 928 0.02 N35S, H49R, L79P, F91S, T107M, G111R V11A, P18F, D23A, L79P, 1725 45249 1.04 913 0.02 830 1.02 33883 0.70 G80D, V95A, T107M P18S, K70R, L79P, F91S, 1726 16180 0.37 793 0.02 854 1.05 1182 0.02 G111R P18T, D23A, Q60H, L79P, 1717 175673 4.02 161958 4.26 879 1.08 50981 1.05 M90V, F91S, T107M V9L, V11M, P18S, N35S, 1727 2999 0.07 2315 0.06 893 1.09 925 0.02 S54G, Q62K, L79P, L104M, T107M, V115M V9L, P18Y, V25A, V38G, 1728 138011 3.16 26015 0.68 919 1.13 17970 0.37 M55V, A77T, L79P, M90I, F91S, L104M V10G, P18T, L72Q, L79P, 1729 4253 0.10 1584 0.04 863 1.06 3643 0.07 F91S, T107M P18S, H59R, A76G, R78S, 1730 130622 2.99 79435 2.09 1009 1.24 44493 0.91 L79P V9A, P18S, M36T, S65G, 1731 92503 2.12 989 0.03 886 1.09 7850 0.16 L79P, F91S, L104T, G111R, S112I P18T, S52A, V57A, Q60R, 1732 187338 4.29 10579 0.28 908 1.11 3791 0.08 Q62K, S65C, L79P, F91T, N100Y, T107M V11A, P18F, N35D, A47E, 1733 Little to no protein produced Q62K, L79P, F91S, G99D, T107M, 5114N V11A, P18T, N355, L79P, 1734 218660 5.00 273825 7.20 1269 1.56 69871 1.44 S87T, F91S V9D, V11M, Q12L, P18S, 1735 8693 0.20 790 0.02 852 1.04 1991 0.04 E37V, M55I, Q60R, K70Q, L79P, F91S, L104M, T107M T15S, P18S, Y30H, Q32L, 1736 16213 0.37 2092 0.06 1056 1.29 6994 0.14 Q62R, L79P, F91S, T107M CD155-ECD-Fc 47 43704 1.00 38032 1.00 816 1.00 48638 1.00 (ECD) CD112-IgV 795 1289 824 17819 1172 0.02

TABLE 14A Variant CD112 selected against cognate binding partners. Molecule sequences, binding data, and costimulatory bioactivity data. TIGIT CD112R Mock Anti-CD3 tfxn tfxn CD226 Expi293 IFN-gamma MFI MFI MFI MFI (pg/mL) SEQ (TIGIT (CD112R (CD226 (Mock (Anti-CD3 ID MFI MFI MFI MFI IFN-gamma NO parental parental parental parental parental CD112 mutation(s) (IgV) ratio) ratio) ratio) ratio) ratio) WT CD112 795 210829 1452 265392 1112 676.6 (1.00) (1.00) (1.00) (1.00) (1.00) Y33H, A112V, G117D 796 12948 1552 1368 1241 164.8 (0.06) (1.07) (0.01) (1.12) (0.24) V19A, Y33H, S64G, S80G, 797 48356 1709 2831 1098 G98S, N106Y, A112V (0.23) (1.18) (0.01) (0.99) L32P, A112V 798 191432 1557 11095 1259 390.4 (0.91) (1.07) (0.04) (1.13) (0.58) A95V, A112I 799 238418 1706 51944 1215 282.5 (1.13) (1.17) (0.20) (1.09) (0.42) P28S, A112V 800 251116 1985 153382 1189 503.4 (1.19) (1.37) (0.58) (1.07) (0.74) P27A, T38N, V101A, 801 255803 2138 222822 1399 240.7 A112V (1.21) (1.47) (0.84) (1.26) (0.36) S118F 802 11356 5857 6938 1270 271.7 (0.05) (4.03) (0.03) (1.14) (0.40) R12W, H48Y, F54S, S118F 803 10940 3474 5161 1069 (0.05) (2.39) (0.02) (0.96) R12W, Q79R, S118F 804 2339 7370 1880 1338 447.4 (0.01) (5.08) (0.01) (1.20) (0.66) T113S, S118Y 805 6212 6823 1554 1214 225.1 (0.03) (4.70) (0.01) (1.09) (0.33) S118Y 806 2921 6535 2003 1463 190.4 (0.01) (4.50) (0.01) (1.32) (0.28) N106I, S118Y 807 2750 7729 1815 1222 265.8 (0.01) (5.32) (0.01) (1.10) (0.39) N106I, S118F 808 1841 9944 1529 1308 437.9 (0.01) (6.85) (0.01) (1.18) (0.65) A95T, L96P, S118Y 809 2352 4493 1412 1329 292.4 (0.01) (3.09) (0.01) (1.19) (0.43) Y33H, P67S, N106Y, 810 225015 3259 204434 1296 618.8 A112V (1.07) (2.24) (0.77) (1.17) (0.91) N106Y, A112V 811 6036 1974 15334 1108 409.9 (0.03) (1.36) (0.06) (1.00) (0.61) T18S, Y33H, A112V 812 252647 1347 183181 1412 601.8 (1.20) (0.93) (0.69) (1.27) (0.89) P9S, Y33H, N47S, A112V 813 240467 1418 203608 1361 449.1 (1.14) (0.98) (0.77) (1.22) (0.66) P42S, P67H, A112V 814 204484 1610 188647 1174 530.6 (0.97) (1.11) (0.71) (1.06) (0.78) P27L, L32P, P42S, A112V 815 219883 1963 84319 1900 251.6 (1.04) (1.35) (0.32) (1.71) (0.37) G98D, A112V 816 4879 2369 6100 1729 387.0 (0.02) (1.63) (0.02) (1.55) (0.57) Y33H, S35P, N106Y, 817 250724 1715 94373 1495 516.2 A112V (1.19) (1.18) (0.36) (1.34) (0.76) L32P, P42S, T100A, 818 242675 1742 202567 1748 435.3 A112V (1.15) (1.20) (0.76) (1.57) (0.64) P27S, P45S, N106I, A112V 819 223557 1799 84836 1574 277.5 (1.06) (1.24) (0.32) (1.42) (0.41) Y33H, N47K, A112V 820 251339 1525 199601 1325 483.2 (1.19) (1.05) (0.75) (1.19) (0.71) Y33H, N106Y, A112V 821 297169 1782 258315 1440 485.4 (1.41) (1.23) (0.97) (1.30) (0.72) K78R, D84G, A112V, 822 236662 1638 24850 1345 142.5 F114S (1.12) (1.13) (0.09) (1.21) (0.21) F54L, A112V 823 14483 1617 2371 1353 352.8 (0.07) (1.11) (0.01) (1.22) (0.52) Y33H, A112V 824 98954 1216 1726 1298 (0.47) (0.84) (0.01) (1.17) A95V, A112V 825 168521 2021 200789 1459 412.9 (0.80) (1.39) (0.76) (1.31) (0.61) R12W, A112V 826 135635 1582 23378 1412 165.8 (0.64) (1.09) (0.09) (1.27) (0.24) A112V 832 213576 1986 151900 1409 211.4 (1.01) (1.37) (0.57) (1.27) (0.31) Y33H, A112V 824 250667 1628 230578 1216 612.7 (1.19) (1.12) (0.87) (1.09) (0.91) R12W, P27S, A112V 827 3653 1308 9105 1051 (0.02) (0.90) (0.03) (0.94) Y33H, V51M, A112V 828 218698 1384 195450 1170 709.4 (1.04) (0.95) (0.74) (1.05) (1.05) Y33H, A112V, S118T 829 219384 1566 192645 1313 396.3 (1.04) (1.08) (0.73) (1.18) (0.59) Y33H, V101A, A112V, 830 5605 1582 5079 1197 P115S (0.03) (1.09) (0.02) (1.08) H24R, T38N, D43G, 831 227095 1537 229311 1336 858.6 A112V (1.08) (1.06) (0.86) (1.20) (1.27) A112V 832 4056 1356 10365 986 (0.02) (0.93) (0.04) (0.89) P27A, A112V 833 193537 1531 230708 3084 355.1 (0.92) (1.05) (0.87) (2.77) (0.52) A112V, S118T 834 233173 1659 121817 845 533.3 (1.11) (1.14) (0.46) (0.76) (0.79) R12W, A112V, M122I 835 235935 1463 217748 1350 528.0 (1.12) (1.01) (0.82) (1.21) (0.78) Q83K, N106Y, A112V 836 205948 2042 234958 1551 481.4 (0.98) (1.41) (0.89) (1.39) (0.71) R12W, P27S, A112V, 837 11985 2667 12756 1257 334.4 S118T (0.06) (1.84) (0.05) (1.13) (0.49) P28S, Y33H, A112V 838 4711 1412 3968 955 (0.02) (0.97) (0.01) (0.86) P27S, Q90R, A112V 839 3295 1338 6755 1048 (0.02) (0.92) (0.03) (0.94) L15V, P27A, A112V, 840 209888 1489 84224 1251 512.3 S118T (1.00) (1.03) (0.32) (1.13) 0.76) Y33H, N106Y, T108I, 841 Not tested A112V Y33H, P56L, V75M, 842 Not tested V101M, A112V

TABLE 14B Additional CD112 Variants and Binding Data. TIGIT CD226 CD112R CD96 SEQ Fold MFI Fold MFI Fold MFI Fold ID MFI Increase at Increase at Increase at Increase CD112 NO 100 to WT 100 to WT 100 to WT 100 to WT Mutation(s) (IgV) nM IgV nM IgV nM IgV nM IgV S118F 802 1763 0.02 1645 0.08 2974 0.61 1659 0.19 N47K, Q79R, 925 1738 0.02 1689 0.09 2637 0.54 1647 0.19 S118F Q40R, P60T, 926 4980 0.06 1608 0.08 2399 0.50 2724 0.32 A112V, S118T F114Y, S118F 927 110506 1.34 7325 0.37 1502 0.31 1553 0.18 N106I, S118Y 807 1981 0.02 1700 0.09 2394 0.49 1582 0.19 S118Y 806 101296 1.23 9990 0.50 1429 0.30 1551 0.18 Y33H, K78R, 928 2276 0.03 2115 0.11 3429 0.71 2082 0.24 S118Y N106I, S118F 808 1875 0.02 1675 0.08 2365 0.49 1662 0.19 R12W, A46T, 929 3357 0.04 1808 0.09 1664 0.34 4057 0.48 K66M, Q79R, N106I, T113A, S118F Y33H, A112V, 930 3376 0.04 2886 0.15 3574 0.74 3685 0.43 S118F R12W, Y33H, 931 100624 1.22 24513 1.24 1490 0.31 2060 0.24 N106I, S118F L15V, Q90R, 932 5791 0.07 4169 0.21 2752 0.57 4458 0.52 S118F N47K, D84G, 933 3334 0.04 2819 0.14 2528 0.52 3498 0.41 N106I, S118Y L32P, S118F 934 3881 0.05 2506 0.13 2659 0.55 2518 0.29 Y33H, Q79R, 935 Low to no protein produced A112V, S118Y T18A, N106I, 936 84035 1.02 10208 0.52 1585 0.33 1590 0.19 S118T L15V, Y33H, 937 Low to no protein produced N106Y, A112V, S118F V37M, S118F 938 96986 1.18 2523 0.13 1985 0.41 1849 0.22 N47K, A112V, 939 1980 0.02 1859 0.09 2733 0.56 1825 0.21 S118Y A46T, A112V 940 4224 0.05 4685 0.24 3288 0.68 4273 0.50 P28S, Y33H, 941 6094 0.07 2181 0.11 1891 0.39 3021 0.35 N106I, S118Y P30S, Y33H, 942 2247 0.03 2044 0.10 1796 0.37 2658 0.31 N47K, V75M, Q79R, N106I, S118Y V19A, N47K, 943 2504 0.03 2395 0.12 2174 0.45 2852 0.33 N106Y, K116E, S118Y Q79R, T85A, 944 2192 0.03 1741 0.09 2367 0.49 1620 0.19 A112V, S118Y Y33H, A112V 824 20646 0.25 1465 0.07 1794 0.37 2589 0.30

TABLE 14B Additional CD112 Variants and Binding Data. TIGIT CD226 CD112R CD96 SEQ Fold Fold Fold Fold ID MFI at Increase MFI at Increase MFI at Increase MFI at Increase CD112 NO 100 to WT 100 to WT 100 to WT 100 to WT Mutation(s) (IgV) nM IgV nM IgV nM IgV nM IgV V101M, N106I, 945 55274 0.67 6625 0.33 1357 0.28 1494 0.17 S118Y Y33H, Q79R, 946 6095 0.07 1760 0.09 2393 0.49 3033 0.36 N106I, A112V, S118T Q79R, A112V 947 1571 0.02 1490 0.08 2284 0.47 1326 0.16 Y33H, A46T, 948 90813 1.10 15626 0.79 1298 0.27 3571 0.42 Q79R, N106I, S118F A112V, G121S 949 95674 1.16 19992 1.01 1252 0.26 4005 0.47 Y33H, Q79R, 950 36246 0.44 2118 0.11 1970 0.41 3250 0.38 N106I, S118Y Y33H, N106I, 951 47352 0.57 4217 0.21 2641 0.55 1488 0.17 A112V Y33H, A46T, 952 14413 0.17 1596 0.08 2335 0.48 1441 0.17 V101M, A112V, S118T L32P, L99M, 953 3056 0.04 1791 0.09 2210 0.46 2000 0.23 N106I, S118F L32P, T108A, 954 104685 1.27 4531 0.23 2308 0.48 1518 0.18 S118F A112V 832 4937 0.06 1903 0.10 1646 0.34 3011 0.35 R12W, Q79R, 955 55539 0.67 6918 0.35 1386 0.29 1740 0.20 A112V Y33H, N106Y, 956 2786 0.03 2517 0.13 1787 0.37 2023 0.24 E110G, A112V Y33H, N106I, 957 1967 0.02 1579 0.08 2601 0.54 1517 0.18 S118Y Q79R, S118F 958 82055 1.00 7582 0.38 1298 0.27 1970 0.23 Y33H, Q79R, 959 21940 0.27 1632 0.08 1141 0.24 18423 2.16 G98D, V101M, A112V N47K, T81S, 960 6889 0.08 1311 0.07 1303 0.27 1145 0.13 V101M, A112V, S118F G82S, S118Y 961 4267 0.05 1938 0.10 2140 0.44 2812 0.33 Y33H, A112V, 962 14450 0.18 1532 0.08 2353 0.49 3004 0.35 S118Y Y33H, N47K, 963 70440 0.85 3557 0.18 1447 0.30 1679 0.20 Q79R, N106Y, A112V Y33H, S118T 964 113896 1.38 17724 0.89 1252 0.26 5001 0.59 R12W, Y33H, 965 3376 0.04 2727 0.14 2047 0.42 2339 0.27 Q79R, V101M, A112V S118F 802 2685 0.03 1864 0.09 2520 0.52 1566 0.18 Wildtype 795 82414 1.00 19803 1.00 4842 1.00 8541 1.00 CD112-IgV Fc CD112 ECD-Fc 48 29157 0.35 8755 0.44 1107 0.23 1103 0.13 (ECD) Anti-hFc PE — 1383 0.02 1461 0.07 1358 0.28 1468 0.17

TABLE 14C Additional CD112 Variants and Binding Data. TIGIT CD226 CD112R CD96 SEQ Fold MFI Fold MFI Fold MFI Fold ID MFI Increase at Increase at Increase at Increase CD112 NO 20 to WT 20 to WT 20 to WT 20 to WT Mutation(s) (IgV) nM IgV nM IgV nM IgV nM IgV N106I, S118Y 807 1288 0.04 1334 0.12 6920 4.16 1102 0.44 Y33H, Q83K, 1503 115690 3.31 10046 0.93 1128 0.68 2053 0.82 A112V, S118T R12W, Q79R, 804 1436 0.04 1296 0.12 6546 3.93 1046 0.42 S118F V29M, Y33H, 1504 Not tested N106I, S118F Y33H, A46T, 1505 111256 3.18 14974 1.39 1148 0.69 3333 1.34 A112V Y33H, Q79R, 1506 1483 0.04 1326 0.12 7425 4.46 1138 0.46 S118F Y33H, N47K, 1507 1338 0.04 1159 0.11 1516 0.91 1140 0.46 F74L, S118F R12W, V101M, 1508 1378 0.04 1249 0.12 5980 3.59 1182 0.47 N106I, S118Y A46T, V101A, 1509 1359 0.04 1199 0.11 6729 4.04 1173 0.47 N106I, S118Y Y33H, N106Y, 821 113580 3.25 17771 1.65 1207 0.72 2476 0.99 A112V N106Y, A112V, 1510 Not tested S118T S76P, T81I, 1511 Not tested V101M, N106Y, A112V, S118F N106Y, A112V 811 29015 0.83 2760 0.26 1159 0.70 1639 0.66 P9R, L21V, 5112 1920 0.05 1218 0.11 1107 0.66 1074 0.43 P22L, I34M, S69F, F74L, A87V, A112V, L125A Y33H, V101M, 1513 126266 3.61 24408 2.27 1150 0.69 4535 1.82 A112V N106I, S118F 808 1776 0.05 1385 0.13 9058 5.44 1370 0.55 V29A, L32P, 1514 1265 0.04 1148 0.11 5057 3.04 1194 0.48 S118F A112V 832 69673 1.99 6387 0.59 1140 0.68 1214 0.49 Y33H, V101M, 1513 133815 3.83 24992 2.32 1184 0.71 6338 2.54 A112V P285, Y33H, 941 2745 0.08 1689 0.16 6625 3.98 1978 0.79 N106I, S118Y Y33H, V101M, 1515 118654 3.40 21828 2.03 1253 0.75 3871 1.55 N106I, A112V R12W, Y33H, 1516 171390 4.91 5077 0.47 1124 0.68 2636 1.06 N47K, Q79R, S118Y A112V, S118T 834 103203 2.95 15076 1.40 1155 0.69 1426 0.57 Y33H, A46T, 1517 141859 4.06 29436 2.74 1184 0.71 5760 2.31 A112V, S118T Y33H, A112V, 1518 5161 0.15 1734 0.16 1184 0.71 1249 0.50 F114L, S118I A112V 832 78902 2.26 6224 0.58 1114 0.67 1181 0.47 Y33H, T38A, 1519 111293 3.19 25702 2.39 1192 0.72 99015 39.69 A46T, V101M, A112V Q79R, A112V 947 96674 2.77 7264 0.67 1130 0.68 1216 0.49 Y33H, N106I, 957 5720 0.16 1453 0.14 6543 3.93 1248 0.50 S118Y P285, Y33H, 1520 22393 0.64 1378 0.13 1550 0.93 19174 7.68 S69P, N106I, A112V, S118Y Y33H, P42L, 1521 214116 6.13 13878 1.29 1315 0.79 4753 1.91 N47K, V101M, A112V Y33H, N47K, 1522 6719 0.19 1319 0.12 1305 0.78 1278 0.51 F745, Q83K, N1061, F111L, A112V, 5118T Y33H, A112V, 1523 184794 5.29 10204 0.95 1269 0.76 4321 1.73 S118T, V119A Y33H, N106I, 1524 6872 0.20 1591 0.15 2308 1.39 2796 1.12 A112V, S118F Y33H, K66M, 1525 1724 0.05 1259 0.12 6782 4.07 1197 0.48 S118F, W124L S118F 802 1325 0.04 1213 0.11 7029 4.22 1135 0.46 N1061, A112V 811 111342 3.19 4241 0.39 1546 0.93 1178 0.47 Y33H, A112V 824 177926 5.09 13761 1.28 1152 0.69 3117 1.25 WT CD112 IgV 795 34932 1.00 10762 1.00 1665 1.00 2495 1.00 WT CD112-Fc 48 28277 0.81 8023 0.75 1253 0.75 1064 0.43 ECD (ECD) Anti-huFc PE — 1138 0.03 1006 0.09 1010 0.61 1062 0.43

TABLE 15A Selected PD-L1 variants and binding data. Binding to Jurkat/PD-1 Cells SEQ Fold increase ID MFI over wildtype NO at PD-L1 PD-L1 Mutation(s) (IgV) 50 nM IgV-Fc K28N, M41V, N45T, H51N, K57E 1389 12585 2.4 120L, I36T, N45D, I47T 1390 3119 0.6 120L, M41K, K44E 1391 9206 1.8 P6S, N4ST, N78I, I83T 1392 419 0.1 N78I 1393 2249 0.4 M41K, N78I 1394 Little or no protein produced N17D, N45T, V50A, D72G 1400 Little or no protein produced I20L, F49S 1401 Little or no protein produced N45T, VS0A 1402 23887 4.6 I20L, N45T, N78I 1403 29104 5.6 N45T, N78I 1395 24865 4.7 I20L, N4ST 1396 24279 4.6 I20L, N45T, VS0A 1404 34158 6.5 N45T 1397 6687 1.3 M41K 1398 5079 1.0 M41V, N45T 1405 Little or no protein produced M41K, N45T 1406 Little or no protein produced A33D, S75P, D85E 1407 685 0.1 M18I, M41K, D43G, H51R, N781 1408 20731 4.0 V11E, 120L, I36T, N45D, H60R, S75P 1409 3313 0.6 A33D, V50A 1410 Little or no protein produced S16G, A33D, K71E, S75P 1411 Little or no protein produced E27G, N4ST, M97I 1412 881 0.2 E27G, N45T, K57R 1413 5022 1.0 A33D, E53V 1414 650 0.1 D43G, N45D, V58A 1415 63960 12.2 E40G, D43V, N45T, V50A 1416 809 0.2 Y14S, K28E, N45T 1417 16232 3.1 A33D, N78S 1418 1725 0.3 A33D, N781 1419 8482 1.6 A33D, N45T 1420 17220 3.3 A33D,N45T, N78I 1421 Little or no protein produced E27G, N45T, V50A 1422 25267 4.8 N45T, V50A, N78S 1423 28572 5.4 N451, V50A 1402 18717 3.6 I20L, N45T, V110M 1424 464 0.1 I20L, I36T, N45T, V50A 1425 7658 1.5 N45T, L74P, S75P 1426 5251 1.0 N45T, S75P 1427 12200 2.3 S75P, K106R 1428 388 0.1 S75P 1429 1230 0.2 A33D, S75P 1430 306 0.1 A33D, S75P, D104G 1431 251 0.0 A33D, S75P 1430 1786 0.3 I20L, E27G, N45T, V50A 1433 29843 5.7 I20L, E27G, D43G, N45D, V58A, N78I 1434 69486 13.3 I20L, D43G, N45D, V58A, N78I 1435 72738 13.9 I20L, A33D, D43G, N45D, V58A, N78I 1436 80205 15.3 I20L, D43G, N45D, N78I 1437 67018 12.8 E27G, N45T, V50A, N78I 1438 30677 5.9 N45T, V50A, N78I 1439 32165 6.1 V11A, I20L, E27G, D43G, N45D, H51Y, 1440 73727 14.1 S99G I20L, E27G, D43G, N45T, V50A 1441 36739 7.0 I20L, K28E, D43G, N45D, V58A, Q89R, 1442 80549 15.4 I20L, I36T, N45D 1443 16870 3.2 I20L, K28E, D43G, N45D, E53G, V58A, 1444 139 0.0 N78I A33D, D43G, N45D, V58A, S75P 1445 58484 11.2 K23R, D43G, N45D 1446 67559 12.9 I20L, D43G, N45D, V58A, N78I, D90G, 1447 259 0.0 G101D D43G, N45D, L56Q, V58A, G101G- 1448 88277 16.8 ins(G101GG) I20L, K23E, D43G, N45D, V58A, N78I 1449 89608 17.1 I20L, K23E, D43G, N45D, V50A, N78I 1450 88829 16.9 T19I, E27G, N45I, V50A, N78I, M97K 1451 25496 4.9 120L, M41K, D43G, N45D 1452 599 0.1 K23R, N45T, N78I 1453 84980 16.2 Full length PD-L1 Fc — 18465 3.5 Wild type PD-L1 IgV 1454 5243 1.0 Anti-PD-1 monoclonal antibody (niVolumab) — 79787 15.2 Human IgG — 198 0.0

TABLE 15B Flow Binding to Cells Expressing PD-1 or CD80 PD-1 CD80 Fold Fold SEQ Change Change ID MFI Compared MFI Compared NO at to WT at to WT PD-L1 Mutation(s) (ECD) 20 nM PD-L1 20 nM PD-L1 K57R, S99G 1813 2953 0.9 16253 121.3 K57R, S99G, F189L 1814 1930 0.6 12906 96.3 M18V, M97L, F193S, R195G, E200K, 1815 69 0.0 241 1.8 H202Q I36S, M41K, M97L, K144Q, R195G, 1816 3498 1.1 68715 512.8 E200K, H202Q, L206F C22R, Q65L, L124S, K144Q, R195G, 1817 Little or no protein produced E200N, H202Q, T221L M18V, I98L, L124S, P198T, L206F 1818 2187 0.7 143 1.1 S99G, N117S, I148V, K171R, R180S 1819 Little or no protein produced I36T, M97L, A103V, Q155H 1820 120 0.0 128 1.0 K28I, S99G 1821 830 0.3 693 5.2 R195S 1822 3191 1.0 138 1.0 A79T, S99G, T185A, R195G, E200K, 1823 1963 0.6 643 4.8 H202Q, L206F K57R, S99G, L124S, K144Q 1824 2081 0.7 14106 105.3 K57R, S99G, R195G 1825 2479 0.8 10955 81.8 D55V, M97L, S99G 1826 11907 3.8 71242 531.7 E27G, I36T, D55N, M97L, K111E 1827 1904 0.6 88724 662.1 E54G, M97L, S99G 1828 8414 2.7 51905 387.4 G15A, I36T, M97L, K111E, H202Q 1829 112 0.0 13530 101.0 G15A, I36T, V129D 1830 114 0.0 136 1.0 G15A, I36T, V129D, R195G 1831 125 0.0 134 1.0 G15A, V129D 1832 2075 0.7 128 1.0 I36S, M97L 1833 3459 1.1 44551 332.5 I36T, D55N, M97L, K111E, A204T 1834 265 0.1 62697 467.9 I36T, D55N, M97L, K111E, V129A, 1835 393 0.1 72641 542.1 F173L I36T, D55S, M97L, K111E, I148V, 1836 94 0.0 30704 229.1 R180S I36T, G52R, M97L, V112A, K144E, 1837 81 0.0 149 1.1 V175A, P198T I36T, I46V, D55G, M97L, K106E, 1838 69 0.0 190 1.4 K144E, T185A, R195G I36T, I83T, M97L, K144E, P198T 1839 62 0.0 6216 46.4 I36T, M97L, K111E 1840 Little or no protein produced I36T, M97L, K144E, P198T 1841 197 0.1 40989 305.9 I36T, M97L, Q155H, F193S, N201Y 1842 69 0.0 1251 9.3 I36T, M97L, V129D 1843 523 0.2 50905 379.9 L35P, I36S, M97L, K111E 1844 190 0.1 155 1.2 M181, I36T, E53G, M97L, K144E, 1845 104 0.0 47358 353.4 E199G, V207A M18T, I36T, D55N, M97L, K111E 1846 138 0.0 71440 533.1 M18V, M97L, T176N, R195G 1847 1301 0.4 45300 338.1 M97L, S99G 1848 12906 4.1 81630 609.2 N17D, M97L, S99G 1849 10079 3.2 73249 546.6 S99G, T185A, R195G, P198T 1850 2606 0.8 22062 164.6 V129D, H202Q 1851 2001 0.6 219 1.6 V129D, P198T 1852 3245 1.0 152 1.1 V129D, T150A 1853 1941 0.6 142 1.1 V93E, V129D 1854 1221 0.4 150 1.1 Y10F, M18V, S99G, Q138R, T203A 1855 70 0.0 412 3.1 WT PD-L1 (IgV + IgC) Fc 1812 3121 1.0 134 1.0 CTLA4-Fc — 59 N/A 199670 N/A Anti-PD1 mAb — 31482 N/A 134 N/A Fc Control 1189 59 N/A 132 N/A

TABLE 15C Additional Affinity-Matured IgSF Domain-Containing Molecules SEQ ID NO PD-L1 Mutation(s) (ECD) N45D 1945 K160M, R195G 1946 N45D, K144E 1947 N45D, P198S 1948 N45D, P198T 1949 N45D, R195G 1950 N45D, R195S 1951 N45D, S131F 1952 N45D, V58D 1953 V129D, R195S 1954 I98T, F173Y, L196S 1955 N45D, E134G, L213P 1956 N45D, F173I, S177C 1957 N45D, I148V, R195G 1958 N45D, K111T, R195G 1959 N45D, N113Y, R195S 1960 N45D, N165Y, E170G 1961 N45D, Q89R, I98V 1962 N45D, S131F, P198S 1963 N45D, S75P, P198S 1964 N45D, V50A, R195T 1965 E27D, N45D, T183A, I188V 1966 F173Y, T183I, L196S, T203A 1967 K23N, N45D, S75P, N120S 1968 N45D, G102D, R194W, 1969 R195G N45D, G52V, Q121L, P198S 1970 N45D, I148V, R195G, N201D 1971 N45D, K111T, T183A, I188V 1972 N45D, Q89R, F189S, P198S 1973 N45D, S99G, C137R, V207A 1974 N45D, T163I, K167R, R195G 1975 N45D, T183A, T192S, R194G 1976 N45D, V50A, I119T, K144E 1977 T19A, N45D, K144E, R195G 1978 V11E, N45D, T130A, P198T 1979 V26A, N45D, T163I, T185A 1980 K23N, N45D, L124S, K167T, 1981 R195G K23N, N45D, Q73R, T163I 1982 K28E, N45D, W149R, S158G, 1983 P198T K28R, N45D, K57E, I98V, 1984 R195S K28R, N45D, V129D, T163N, 1985 R195T M41K, D43G, N45D, R64S, 1986 R195G M41K, D43G, N45D, R64S, 1987 S99G N45D, R68L, F173L, D197G, 1988 P198S N45D, V50A, I148V, R195G, 1989 N201D M41K, D43G, K44E, N45D, 1990 R195G, N201D N45D, V50A, L124S, K144E, 1991 L179P, R195G

Example 9 Generation of Stacked Molecules Containing Different Affinity-Modified Domains

This Example describes further immunomodulatory proteins that were generated as multi-domain stack constructs containing at least two different affinity modified IgV domains from identified variant PD-L2 polypeptides and identified variant CD155 polypeptides described above. Specifically, the exemplary variant PD-L2 IgV H15Q/T47A/K65R/S67L/Q82R/V89D (SEQ ID NO:328) and the exemplary variant CD155 IgV molecule P18S/S65W/S67A/L104Q/G111R (SEQ ID NO:1598) were linked together and fused to an Fc in various configurations. Stack constructs were obtained as geneblocks (Integrated DNA Technologies, Coralville, Iowa) that either encoded the entire chain or they were generated by obtaining geneblocks that encode PDL2-CD155 in its various configurations for subsequent Gibson assembly into Fc fusion vector using a Gibson assembly kit (New England Biolabs). Homodimeric and heterodimeric stacks were generated in various configurations as summarized in FIG. 5A and as follows.

Homodimeric stack constructs were generated containing identical Fc subunits in which the variant PD-L2 IgV and variant CD155 IgV were variously linked to the N- or C-terminus of a human IgG1 Fc region via a 2×GGGS (SEQ ID NO:264) or 3×GGGGS (SEQ ID NO: 263) peptide linker. In this study, the exemplary IgG1 Fc subunit is set forth in SEQ ID NO:1189 and contained the mutations L234A, L235E, G237A, E356D and M358L by EU numbering (corresponding to L19A, L20E, G22A, E141D and M143L with reference to wild-type human IgG1 Fc set forth in SEQ ID NO:211). Further, the Fc region contained replacement of the cysteine residues to a serine residue at position 5 (C5 S) compared to the wild-type or unmodified Fc set forth in SEQ ID NO: 211 (corresponding to C220S by EU numbering). In some examples, the exemplary IgG1 Fc set forth in SEQ ID NO: 1189 was used, which contained the above mutations and additionally lacked the C-terminal lysine corresponding to position 232 of the wild-type or unmodified Fc set forth in SEQ ID NO: 211 (corresponding to K447del by EU numbering). Other Fc regions also are suitable for generation of stack molecules. Exemplary generated stacks are set forth below.

The homodimeric variant IgV-stacked-Fc fusion molecules containing various configurations of variant IgV domains from PD-L2 (SEQ ID NO: 328) and CD155 (SEQ ID NO:1598) were expressed and purified substantially as described in Example 5.

The encoding nucleic acid molecule was designed to produce homodimeric stacks in various configurations of sequences in the order shown:

PD-L2/CD155 Stack 1 (SEQ ID NO: 1191): CD155 variant (SEQ ID NO: 1598) - 2xGGGS (SEQ ID NO: 264) -Fc (SEQ ID NO: 1189) - 3x GGGGS (SEQ ID NO: 263) - PD-L2 (SEQ ID NO: 328) PD-L2/CD155 Stack 2 (SEQ ID NO: 1192): PD-L2 (SEQ ID NO: 328) - 2xGGGS (SEQ ID NO: 264) -Fc (SEQ ID NO: 1189) - 3x GGGGS (SEQ ID NO: 263) - CD155 variant (SEQ ID NO: 1598) PD-L2/CD155 Stack 3 (SEQ ID NO: 1193): CD155 variant (SEQ ID NO: 1598) -3x GGGGS (SEQ ID NO: 263) - PD-L2 (SEQ ID NO: 328) - 2xGGGS (SEQ ID NO: 264) -Fc (SEQ ID NO: 1189) PD-L2/CD155 Stack 4 (SEQ ID NO: 1194): PD-L2 (SEQ ID NO: 328) - 3x GGGGS (SEQ ID NO: 263) - CD155 variant (SEQ ID NO: 1598) - 2xGGGS (SEQ ID NO: 264) -Fc (SEQ ID NO: 1189) PD-L2/CD155 Stack 5 (SEQ ID NO: 1195): Fc (SEQ ID NO: 1189) - 3x GGGGS (SEQ ID NO: 263) - CD155 variant (SEQ ID NO: 1598) - 3xGGGS (SEQ ID NO: 263) - PD-L2 (SEQ ID NO: 328) PD-L2/CD155 Stack 6 (SEQ ID NO: 1196): Fc (SEQ ID NO: 1189) - 3x GGGGS (SEQ ID NO: 263) - PD-L2 (SEQ ID NO: 328) - 3xGGGS (SEQ ID NO: 263) - CD155 variant (SEQ ID NO: 1598)

Heterodimeric stacks were generated in two ways. The first way was by co-expression of the variant PD-L2 IgV and/or variant CD155 IgV fused to (1) a first “knob” Fc subunit (set forth in SEQ ID NO:1187 containing the mutations S354C and T366W by EU numbering, corresponding to S139C and T151W with reference to wild-type human IgG1 Fc set forth in SEQ ID NO:211); and (2) a second “hole” Fc subunit (set forth in SEQ ID NO:1188, containing the mutations Y349C, T366S, L368A and Y407V by EU numbering, corresponding to Y134C, T151S, L153A and Y192V with reference to wild-type human IgG1 Fc set forth in SEQ ID NO:211) for expression of a heterodimeric molecule by “knobs-into-hole” engineering. In addition, both the knob and hole Fc also contained mutations L19A, L20E, G22A to reduce effector function and contained replacement of the cysteine residue to a serine residue at position 5 (C5 S), each compared to the wild-type or unmodified Fc set forth in SEQ ID NO: 211 (corresponding to C220S, L234A, L235E and G237A by EU numbering, respectively). In a second way, PDL2 and/or CD155 were fused to both knob and hole Fc's to generate stacks where each chain consists of Fc with fused IgV domain(s). For constructs in which the Fc sequence was the N-terminal portion of the sequence, a stuffer sequence HMSSVSAQ (SEQ ID NO:1190) was added immediately preceding the Fc sequence.

The heterodimeric variant IgV-stacked-Fc fusion molecules containing various configurations of variant IgV domains from PD-L2 (SEQ ID NO: 328) and CD155 (SEQ ID NO:1598) were expressed and purified substantially as described in Example 5. For each stack, the encoding nucleic acid molecule of the knob and hole were designed to produce heterodimeric stacks in various configurations with sequences in the order shown:

PD-L2/CD155 Stack 7 containing (1) knob Fc fusion (SEQ ID NO: 1197): CD155 variant (SEQ ID NO: 1598) - 2xGGGS (SEQ ID NO: 264) - knob Fc (SEQ ID NO: 1187) - 3x GGGGS (SEQ ID NO: 263) - PD-L2 (SEQ ID NO: 328)  and (2) hole Fc (SEQ ID NO: 1188 plus N-terminal HMSSVSAQ set forth in SEQ ID NO: 1190) PD-L2/CD155 Stack 8 containing (1) knob Fc fusion (SEQ ID NO: 1198): PD-L2 (SEQ ID NO: 328) - 2xGGGS (SEQ ID NO: 264) - knob Fc (SEQ ID NO: 1187) - 3x GGGGS (SEQ ID NO: 263) - CD155 variant (SEQ ID NO: 1598) and (2) hole Fc (SEQ ID NO: 1188 plus N-terminal HMSSVSAQ set forth in SEQ ID NO: 1190) PD-L2/CD155 Stack 9 containing (1) knob Fc fusion (SEQ ID NO: 1199): CD155 variant (SEQ ID NO: 1598) - 3x GGGGS (SEQ ID NO: 263) - CD155 variant (SEQ ID NO: 1598) - 2xGGGS (SEQ ID NO: 264) - knob Fc (SEQ ID NO: 1187) - 3x GGGGS (SEQ ID NO: 263) - PD-L2 (SEQ ID NO: 328) - 3x GGGGS (SEQ ID NO: 263) - PD-L2 (SEQ ID NO: 328); and (2) hole Fc (SEQ ID NO: 1188 plus N-terminal HMSSVSAQ set forth in SEQ ID NO: 1190) PD-L2/CD155 Stack 10 containing (1) knob Fc fusion (SEQ ID NO: 1200): PD-L2 (SEQ ID NO: 328) - 3x GGGGS (SEQ ID NO: 263) - PD-L2 (SEQ ID NO: 328) - 2xGGGS (SEQ ID NO: 264) - knob Fc (SEQ ID NO: 1187) - 3x GGGGS (SEQ ID NO: 263) - CD155 variant (SEQ ID NO: 1598) - 3x GGGGS (SEQ ID NO: 263) - CD155 variant (SEQ ID NO: 1598); and (2) hole Fc (SEQ ID NO: 1188 plus N-terminal HMSSVSAQ set forth in SEQ ID NO: 1190) PD-L2/CD155 Stack 11 containing (1) knob fc fusion (SEQ ID NO: 1201): CD155 variant (SEQ ID NO: 1598) - 3x GGGGS (SEQ ID NO: 263) - CD155 variant (SEQ ID NO: 1598) - 2xGGGS (SEQ ID NO: 264) -knob Fc (SEQ ID NO: 1187); and (2) hole Fc fusion (SEQ ID NO: 1202): PD-L2 (SEQ ID NO: 328) - 3x GGGGS (SEQ ID NO: 263) - PD-L2 (SEQ ID NO: 328) - 2xGGGS (SEQ ID NO: 264) - hole Fc (SEQ ID NO: 1188) PD-L2/CD155 Stack 12 containing (1) knob fc fusion (SEQ ID NO: 1203): knob Fc (SEQ ID NO: 1187 plus N-terminal HMSSVSAQ set forth in SEQ ID NO: 1190) - 3x GGGGS (SEQ ID NO: 263) - CD155 variant (SEQ ID NO: 1598) - 3x GGGGS (SEQ ID NO: 263) CD155 variant (SEQ ID NO: 1598); and (2) hole Fc (SEQ ID NO: 1204): hole Fc (SEQ ID NO: 1188 plus N-terminal HMSSVSAQ set forth in SEQ ID NO: 1190) - 3x GGGGS (SEQ ID NO: 263) - PD-L2 (SEQ ID NO: 328) - 3x GGGGS (SEQ ID NO: 263) - PD-L2 (SEQ ID NO: 328) PD-L2/CD155 Stack 13 containing (1) knob Fc fusion (SEQ ID NO: 1199): CD155 variant (SEQ ID NO: 1598) - 3x GGGGS (SEQ ID NO: 263) - CD155 variant (SEQ ID NO: 1598) - 2xGGGS (SEQ ID NO: 264) - knob Fc (SEQ ID NO: 1187) - 3x GGGGS (SEQ ID NO: 263) - PD-L2 (SEQ ID NO: 328) - 3x GGGGS (SEQ ID NO: 263) - PD-L2 (SEQ ID NO: 328); and (2) hole Fc (SEQ ID NO: 1204): hole Fc (SEQ ID NO: 1188 plus N-terminal HMSSVSAQ set forth in SEQ ID NO: 1190) - 3x GGGGS (SEQ ID NO: 263) - PD-L2 (SEQ ID NO: 328) - 3x GGGGS (SEQ ID NO: 263) - PD-L2 (SEQ ID NO: 328) PD-L2/CD155 Stack 14 containing (1) knob Fc fusion (SEQ ID NO: 1199): CD155 variant (SEQ ID NO: 1598) - 3x GGGGS (SEQ ID NO: 263) - CD155 variant (SEQ ID NO: 1598) - 2xGGGS (SEQ ID NO: 264) - knob Fc (SEQ ID NO: 1187) - 3x GGGGS (SEQ ID NO: 263) - PD-L2 (SEQ ID NO: 328) - 3x GGGGS (SEQ ID NO: 263) - PD-L2 (SEQ ID NO: 328); and (2) hole Fc fusion (SEQ ID NO: 1202): PD-L2 (SEQ ID NO: 328) - 3x GGGGS (SEQ ID NO: 263) - PD-L2 (SEQ ID NO: 328) - 2xGGGS (SEQ ID NO: 264) - hole Fc (SEQ ID NO: 1188) PD-L2/CD155 Stack 15 containing (1) knob Fc fusion (SEQ ID NO: 1200): PD-L2 (SEQ ID NO: 328) - 3x GGGGS (SEQ ID NO: 263) - PD-L2 (SEQ ID NO: 328) - 2xGGGS (SEQ ID NO: 264) - knob Fc (SEQ ID NO: 1187) - 3x GGGGS (SEQ ID NO: 263) - CD155 variant (SEQ ID NO: 1598) - 3x GGGGS (SEQ ID NO: 263) - CD155 variant (SEQ ID NO: 1598); and (2) hole Fc fusion (SEQ ID NO: 1202): PD-L2 (SEQ ID NO: 328) - 3x GGGGS (SEQ ID NO: 263) - PD-L2 (SEQ ID NO: 328) 2xGGGS (SEQ ID NO: 264) - hole Fc (SEQ ID NO: 1188) PD-L2/CD155 Stack 16 containing (1) knob Fc fusion (SEQ ID NO: 1200): PD-L2 (SEQ ID NO: 328) - 3x GGGGS (SEQ ID NO: 263) - PD-L2 (SEQ ID NO: 328) - 2xGGGS (SEQ ID NO: 264) - knob Fc (SEQ ID NO: 1187) - 3x GGGGS (SEQ ID NO: 263) - CD155 variant (SEQ ID NO: 1598) - 3x GGGGS (SEQ ID NO: 263) - CD155 variant (SEQ ID NO: 1598); and (2) hole Fc (SEQ ID NO: 1204): hole Fc (SEQ ID NO: 1188 plus N-terminal HMSSVSAQ set forth in SEQ ID NO: 1190) - 3x GGGGS (SEQ ID NO: 263) - PD-L2 (SEQ ID NO: 328) - 3x GGGGS (SEQ ID NO: 263) - PD-L2 (SEQ ID NO: 328)

Example 10 Generation of Secreted Immunomodulatory Protein

To generate a PD-L2 secreted immunomodulatory protein (SIP), DNA encoding exemplary SIPs was obtained as gene blocks from Integrated DNA Technologies (Coralville, USA) and then cloned by Gibson assembly (New England Biolabs Gibson assembly kit) into a modified version of pRRL vector (Dull et al., (1998) J Virol, 72(11): 8463-8471) between restriction sites downstream of MND promoter to remove GFP. Exemplary SIP constructs were generated to encode a protein set forth in SEQ ID NO: 1254-1257, including the signal peptide. In this exemplary Example, the constructs were generated to additionally include a tag moiety. The gene blocks had the following structure in order: 39 base pair overlap with pRRL prior to first restriction site-first restriction site-GCCGCCACC (Kozak); complete ORF encoding PD-L2 IgV wildtype amino acid sequence set forth in SEQ ID NO:115 or variant PD-L2 IgV set forth in SEQ ID NO:316 (H15Q,V31M,S67L,Q82R,V89D), SEQ ID NO:328 (H15Q,T47A,K65R,S67L,Q82R,V89D) or SEQ ID NO: 342 (H15Q,T47A,S67L,R76G,Q82R,V89D), also including in all cases the PD-L2 signal peptide MIFLLLMLSLELQLHQIAA as set forth in SEQ ID NO: 1251; DNA encoding Avitag as set forth in SEQ ID NO:1252 (GLNDIFEAQKIEWHE); DNA encoding His tag as set forth in SEQ ID NO: 1253 (HHHHHH); TAA stop codon; second restriction site-41 base pair overlap with pRRL beyond second restriction site.

To prepare lentiviral vectors, 3×10⁶ HEK293 cells were plated per 100 mm dish. On the next day, 4.5 μg of P-Mix (3 μg of PAX2 and 1.5 μg of pMD2G) was added to 6 μg of DNA encoding the SIPs constructs in a 5 mL polypropylene tube. Diluent buffer (10 mM HEPES/150 mM NaCl pH7.05/1 L TC grade H20) was added to the tube to bring up the total volume of 500 μL. To the diluent DNA (PEI:total DNA 4:1), 424, of PEI (1 μg/μL) was added and mixed by vortexing. The mixture was incubated at room temperature for 10 minutes and cells were prepared by aspirating medium from the dish gently without disturbing the adherent cells, then replaced with 6 mL of Opti-MEM(1×). DNA/PEI mixture was then added to the dish and incubated at 37° C. for 24 hours. After 24 hours, media was aspirated from the dishes and replaced with 10 mL of fresh DMEM media and then incubated at 37° C. Viral supernatant was collected after 48 hours using a syringe attached to a 0.45 μm filter PES to remove cells and debris from the culture (Thermo Scientific Nalgene Syringe Filter). A separate lentiviral vector stock also was prepared encoding an anti-CD19 CAR (containing an anti-CD19 scFv, a hinge and transmembrane domain derived from CD8 and a CD3zeta signaling domain) substantially as described. The exemplary anti-CD19 CAR used is set forth in SEQ ID NO: 2024 (encoded by the sequence in set forth in SEQ ID NO: 2025) containing the scFv set forth in SEQ ID NO:1211, the CD8-derived hinge and transmembrane domain set forth in SEQ ID NO: 266, and the CD3zeta set forth in SEQ ID NO:267

Pan T-cells were transduced with the viral vectors encoding the PD-L2 SIPs. T-cells were thawed and activated with anti-CD3/anti-CD28 beads (Dynal) at a 1:1 ratio. The T-cells (1×10⁶ cells) were mixed with 1 mL total lentiviral vector supernatant containing equal volume (0.5 mL each) of the lentiviral vector supernatant encoding the indicated PD-L2 SIPs and a lentiviral vector supernatant encoding the anti-CD19 CAR. As a control, cells were transduced only with the lentiviral vector encoding the anti-CD19 CAR or were transduced with mock vector control. Transduction was performed in the presence of 10 μg/mL polybrene and 50 IU/mL IL-2. Cells were spun down at 2500 rpm for 60 min at 30° C. After 24 hours, 3 mL of Xvivo15 plus media and IL2 was added to each well. The cells were fed every two days with fresh media and cytokines.

Transduction also was carried out on HEK-293 cells, which were resuspended at 2×10⁵ cells with 1 mL of the lentiviral supernatant encoding the indicated PD-L2 SIPs. To the cells, 3 mL of DMEM media was added and cells were fed every two days with fresh media.

To assess the amount of secreted SIP, a cell-based assay was performed to assess binding of the secretable variant PD-L2 to PD-1. Approximately, 100,000 PD-1+ Jurkat cells were plated per well in the presence of 504, of culture supernatant containing PD-L2 SIP obtained from transduced cells above and incubated at 4° C. for 30 minutes. To generate a standard curve, 50 μL of the respective variant PD-L2 protein was added to PD-1+ Jurkat cells at 10 μg/mL, 3 μg/mL, 1 μg/mL, 0.3 μg/mL, 0.1 μg/mL, and 0 μg/mL and also incubated at 4° C. for 30 minutes. Cells were washed and 50 μL of anti-his-APC were added (1:50) and this was incubated at 4° C. for 30 minutes. Surface bound PD-L2 protein was detected by flow cytometry and the concentration of SIP in the supernatant sample was determined by comparison to the standard curve. As shown in FIGS. 11A and 11B, SIP proteins were detected in the supernatant of transduced T cells and transduced HEK293 cell, but were not detected from supernatant samples from mock transduced or cells transduced without SIPs.

Example 11 Assessment of Proliferation and Bioactivity of Pan T Cells Transduced with PD-L2 SIP

Pan T-cells were transduced essentially as described in Example 10 with the viral vectors encoding the PD-L2 SIPs. T-cells were thawed and activated with anti-CD3/anti-CD28 beads (Dynal) at a 1:1 ratio. The T-cells (1×10⁶ cells) were mixed with 1 mL total lentiviral vector supernatant containing equal volume (0.5 mL each) of the lentiviral vector supernatant encoding the indicated PD-L2 SIPs and a lentiviral vector supernatant encoding the anti-CD19 CAR. As a control, cells were transduced only with the lentiviral vector encoding the anti-CD19 CAR or were transduced with mock vector control. Transduction was performed in the presence of 10 μg/mL polybrene and 50 IU/mL IL-2. Cells were spun down at 2500 rpm for 60 min at 30° C. After 24 hours, 3 mL of Xvivo15 plus media and IL2 was added to each well. The cells were fed every two days with fresh media and cytokines.

At 14 days after activation, cells were re-stimulated with Nalm6 cells that had been transduced with a lenti-viral vector to provide expression of PDL1. Transduced T cells were labeled with Cell Trace Far Red and proliferation was measured by determining the fraction of the cells that showed dilution of the dye. Results for the proliferation studies for T cells transduced with exemplary tested variant PD-L2 SIP are shown in FIG. 12A.

Levels of IFN-gamma released into the supernatant were measured by ELISA on day 5 after re-stimulation. Results for the bioactivity studies for T cells transduced with exemplary tested variant PD-L2 SIP are shown in FIG. 12B. The T cells transduced with PD-L2 variant SIP are identified with reference to the amino acid substitutions in the IgV of PD-L2 with reference to positions corresponding to positions of the unmodified (wildtype) PD-L2 ECD sequence set forth in SEQ ID NO:31. As shown in FIGS. 12A and 12B, proliferation and improved activities to increase immunological activity was observed.

Example 12 Assessment of Binding to Cell-Expressed Counter Structures and Bioactivity of Affinity-Matured IgSF Domain-Containing Stack Molecules

This Example describes Fc-fusion binding studies to show specificity and affinity of exemplary PD-L2/CD155 stack immunomodulatory proteins generated in Example 9 for cognate binding partners. The exemplary PD-L2/CD155 stack immunomodulatory proteins generated in Example 9 also were assessed for Fc-fusion variant protein bioactivity characterization in a human primary T cell in vitro assay.

A. Binding to Cell-Expressed Counter Structure

Binding studies were carried out using Jurkat/IL-2 reporter cells (purchased from Promega Corp. USA) that were transduced to stably express human PD-1 (Jurkat/PD-1 cells), human TIGIT (Jurkat/TIGIT cells) or both PD-1 and TIGIT (Jurkat/PD-1/TIGIT cells). For staining by flow cytometry, 100,000 Jurkat/PD-1, Jurkat/TIGIT, Jurkat/PD-1/TIGIT cells or negative control (Jurkat only) were plated in 96-well round-bottom plates. Cells were spun down and resuspended in staining buffer (PBS (phosphate buffered saline), 1% BSA (bovine serum albumin), and 0.1% sodium azide) for 20 minutes to block non-specific binding. Afterwards, cells were centrifuged again and resuspended in 50 μL staining buffer containing 100 nM to 46 pM of each candidate Fc fusion protein. Primary staining was performed on ice for 90 minutes, before washing cells twice in 200 μL staining buffer. PE-conjugated anti-human Fc (Jackson ImmunoResearch, USA) was diluted 1:150 in 50 μL staining buffer and added to cells and incubated another 30 minutes on ice. Secondary antibody was washed out twice, cells were fixed in 4% formaldehyde/PBS, and samples were analyzed on Intellicyt flow cytometer (Intellicyt Corp., USA).

Mean Fluorescence Intensity (MFI) was calculated with FlowJo Version 10 software (FlowJo LLC, USA). Table 16 sets forth binding activity as measured by the Mean Fluorescence Intensity (MFI) value for binding of 6.25 nM of each stack Fc-fusion molecule to Jurkat/PD-1, Jurkat/TIGIT, and Jurkat/PD-1/TIGIT cells. As shown in Table 16, several stack proteins bind both PD-1 and TIGIT with high affinity.

TABLE 16 Binding of PD-L2/CD155 Stacks to Cell-Expressed Counter Structure Binding to Jurkat Transfectants SEQ MFI at 6.25 nM ID TIGIT + Category Description NO PD1 TIGIT PD1 Homodimers (CD155 IgV)—(G4S)2—Fc—(G4S)3  1191 242 19948 5336 (PD-L2 IgV) (PD-L2 IgV) (G4S)2 Fc (G4S)3  1192 16344 9095 16458 (CD155 IgV) (CD155 IgV) (G4S)3 (PD-L2 IgV)  1193 91 22342 8135 (G4S)2 Fc (PD-L2 IgV) (G4S)3  1194 9218 14516 19256 (CD155 IgV) (G4S)2 Fc Fc (G4S)3 (CD155 IgV)  1195 108 5486 2905 (G4S)3 (PD-L2 IgV) Fc (G4S)3 (PD-L2 IgV)  1196 66 9974 5202 (G4S)3 (CD155 IgV) Heterodimers (CD155 IgV) (G4S)2 knob Fc— 1197 + 1188 107 2544 1512 (G4S)3 (PD-L2 IgV) and hole Fc (PD-L2 IgV) (G4S)2 knob Fc  1198 + 1188 1658 360 6762 (G4S)3 (CD155 IgV) and hole Fc (CD155 IgV) (G4S)3 (CD155 1199 + 1188 289 8677 4371 IgV) (G4S)2 knob Fc (G4S)3  (PD-L2 IgV) (G4S)3 (PD-L2 IgV) and hole Fc (PD-L2 IgV) (G4S)3 (PD-L2 IgV)  1200 + 1188 1594 2554 5509 (G4S)2 knob Fc (G4S)3  (CD155 IgV) (G4S)3 (CD155 IgV) and hole Fc (CD155 IgV) (G4S)3 (CD155 1201 + 1202 1758 9642 9343 IgV) (G4S)2 knob Fc and (PD-L2 IgV) (G4S)3 (PD-L2 IgV)  (G4S)2 hole Fc (CD155 IgV) (G4S)3 (CD155 1199 + 1202 4821 7596 8081 IgV) (G4S)2 knob Fc (G4S)3  (PD-L2 IgV) (G4S)3 (PD-L2 IgV) and (PD-L2 IgV) (G4S)3 (PD-L2 IgV) (G4S)2 hole Fc (CD155 IgV) (G4S)3 (CD155 1199 + 1204 515 8299 4228 IgV) (G4S)2 knob Fc (G4S)3  (PD-L2 IgV) (G4S)3 (PD-L2 IgV) and hole Fc (G4S)3 (PD-L2 IgV)  (G4S)3 (PD-L2 IgV) (PD-L2 IgV) (G4S)3 (PD-L2 IgV)  1200 + 1202 10970 3339 9014 (G4S)2 knob Fc (G4S)3  (CD155 IgV) (G4S)3 (CD155 IgV) and (PD-L2 IgV) (G4S)3  (PD-L2 IgV) (G4S)2 hole Fc (PD-L2 IgV) (G4S)3 (PD-L2 IgV)  1200 + 1204 3785 1475 5989 (G4S)2 knob Fc (G4S)3  (CD155 IgV) (G4S)3 (CD155 IgV) and hole Fc (G4S)3 (PD-L2 IgV) (G4S)3 (PD-L2 IgV) Controls PDL2-IgV 328 22448 73 20280 CD155-IgV 1598 133 22342 4893 Fc Control (homodimer) 1189 44 86 66 Fc Control (heterodimer) 1187 + 1188 42 48 48 Wild Type CD155 full ECD Fc 20 (ECD) 64 4547 249 Wild Type PD-L2 full ECD Fc  4 (ECD) 392 46 314 Irrelevant Ig Control hIgG 41 48 41

B. Assessment of Bioactivity of Affinity-Matured IgSF Domain-Containing Molecules Using Mixed Lymphocyte Reaction (MLR)

Soluble PD-L2/CD155 stack protein bioactivity was tested in a human Mixed Lymphocyte Reaction (MLR). Human primary dendritic cells (DC) were generated by culturing monocytes isolated from PBMC (BenTech Bio, USA) in vitro for 7 days with 50 ng/mL rIL-4 (R&D Systems, USA) and 80 ng/mL rGM-CSF (R&D Systems, USA) in Ex-Vivo 15 media (Lonza, Switzerland). To induce DC maturation, lipopolysaccharide (LPS) (InvivoGen Corp., USA) was added to the DC cultures on day 6 and cells were incubated for an additional 24 hours. Approximately 10,000 matured DC and 100,000 purified allogeneic CD3+ T cells (BenTech Bio, USA) were co-cultured with several concentrations of PD-L2/CD155 stack or control proteins in 96 well round-bottom plates in 200 μL final volume of Ex-Vivo 15 media. Irrelevant human IgG and homodimeric and heterodimeric empty Fc proteins were used as negative controls. As positive controls, PD-L2-Fc (full PD-L2 extracellular domain), wildtype CD155-Fc (full CD155 extracellular domain) were assessed. Variant PD-L2 IgV-Fc fusion proteins were tested at 20 nM. On day 5, IFN-gamma secretion in culture supernatants was analyzed using the Human IFN-gamma Duoset ELISA kit (R&D Systems, USA). Optical density was measured on a BioTek Cytation Multimode Microplate Reader (BioTek Corp., USA) and quantitated against titrated rIFN-gamma standard included in the IFN-gamma Duo-set kit (R&D Systems, USA).

Results for the bioactivity studies for exemplary tested PD-L2/CD155 stack proteins are summarized in Table 17, which sets forth the calculated levels of IFN-gamma in culture supernatants (pg/mL). The sequence identifier (SEQ ID NO) for each stack proteins is set forth in column 3. As shown in Table 17, culture supernatants incubated in the presence of exemplary PD-L2/CD155 stack proteins exhibited altered levels of IFNg production in the MLR assay.

TABLE 17 Bioactivity Data of PD-L2/CD155 Stacks Mixed Lymphocyte Reaction: IFNg at 96 hours Fold Increase SEQ compared ID IFNg to IgG Category Description NO [pg/mL] Control Homodimers (CD155 IgV)  (G4S)2 Fc (G4S)3  1191 3097.0 1.3 (PD-L2 IgV) (PD-L2 IgV) (G4S)2 Fc (G4S)3  1192 3700.3 1.6 (CD155 IgV) (CD155 IgV) (G4S)3 (PD-L2 IgV)  1193 3061.6 1.3 (G4S)2 Fc (PD-L2 IgV) (G4S)3 (CD155 IgV)  1194 2270.0 1.0 (G4S)2 Fc Fc (G4S)3 (CD155 IgV) (G4S)3  1195 2003.5 0.9 (PD-L2 IgV) Fc (G4S)3 (PD-L2 IgV) (G4S)3  1196 2951.0 1.3 (CD155 IgV) Heterodimers (CD155 IgV)—(G4S)2 knobFc  1197 + 1188 2040.4 0.9 (G4S)3 (PD-L2 IgV) and hole Fc (PD-L2 IgV) (G4S)2 knob Fc (G4S)3— 1198 + 1188 3768.6 1.6 (CD155 IgV) and hole Fc (CD155 IgV)—(G4S)3 (CD155 IgV)  1199 + 1188 3549.7 1.5 (G4S)2 knob Fc (G4S)3 (PD-L2 IgV)  (G4S)3 (PD-L2 IgV) and hole Fc (PD-L2 IgV) (G4S)3—(PD-L2 IgV)  1200 + 1188 2568.6 1.1 (G4S)2 knob Fc (G4S)3 (CD155 IgV) (G4S)3 (CD155 IgV) and hole Fc (CD155 IgV) (G4S)3 (CD155 IgV)  1201 + 1202 2572.1 1.1 (G4S)2 knob Fc and (PD-L2 IgV)  (G4S)3 (PD-L2 IgV) (G4S)2 hole Fc (CD155 IgV)—(G4S)3 (CD155 IgV)  1199 + 1202 3216.7 1.4 (G4S)2 knob Fc (G4S)3 (PD-L2 IgV)— (G4S)3 (PD-L2 IgV) and (PD-L2 IgV)— (G4S)3 (PD-L2 IgV) (G4S)2 hole Fc (CD155 IgV)—(G4S)3 (CD155 IgV)  1199 + 1204 2673.4 1.2 (G4S)2 knob Fc (G4S)3 (PD-L2 IgV)— (G4S)3 (PD-L2 IgV) and hole Fc  (G4S)3 (PD-L2 IgV) (G4S)3 (PD-L2 IgV) (PD-L2 IgV) (G4S)3—(PD-L2 IgV)  1200 + 1202 2361.6 1.0 (G4S)2 knob Fc (G4S)3 (CD155 IgV) (G4S)3 (CD155 IgV) and (PD- L2 IgV) (G4S)3 (PD-L2 IgV) (G4S)2— hole Fc (PD-L2 IgV) (G4S)3—(PD-L2 IgV)  1200 + 1204 3311.8 1.4 (G4S)2 knob Fc (G4S)3 (CD155 IgV) (G4S)3 (CD155 IgV) and hole Fc— (G4S)3—(PD-L2 IgV)—(G4S)3—(PD— L2 IgV) Controls PDL2-IgV  328 2367.2 1.0 CD155-IgV 1598 2590.7 1.1 Fc Control (homodimer) 1189 2617.9 1.1 Fc Control (heterodimer) 1187 + 1188 2861.5 1.2 Wild Type CD155 full ECD Fc 20 (ECD) 2481.0 1.1 Wild Type PD-L2 full ECD Fc  4 (ECD) 3298.5 1.4 Irrelevant Ig Control hIgG 2297.6 1.0

The present invention is not intended to be limited in scope to the particular disclosed embodiments, which are provided, for example, to illustrate various aspects of the invention. Various modifications to the compositions and methods described will become apparent from the description and teachings herein. Such variations may be practiced without departing from the true scope and spirit of the disclosure and are intended to fall within the scope of the present disclosure. 

1. A variant PD-L2 polypeptide, comprising an IgV domain or a specific binding fragment thereof, an IgC domain or a specific binding fragment thereof, or both, wherein the variant PD-L2 polypeptide comprises one or more amino acid modifications at one or more positions in an unmodified PD-L2 or a specific binding fragment thereof corresponding to position(s) selected from 15, 89, 82, 67, 2, 12, 13, 18, 23, 24, 28, 31, 32, 33, 36, 37, 39, 44, 45, 46, 47, 48, 58, 59, 65, 69, 71, 72, 73, 74, 75, 76, 77, 85, 86, or 91 with reference to numbering of SEQ ID NO:31.
 2. The variant PD-L2 polypeptide of claim 1, wherein the unmodified PD-L2 comprises (i) the sequence of amino acids set forth in SEQ ID NO:31, (ii) a sequence of amino acids that has at least 95% sequence identity to SEQ ID NO:31; or (iii) a portion of (i) or (ii) comprising an IgV domain or IgC domain or specific binding fragment thereof or both.
 3. The variant PD-L2 polypeptide of claim 1, wherein: the variant PD-L2 comprises up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid modifications compared to the unmodified PD-L2 polypeptide; and/or the variant PD-L2 polypeptide comprises a sequence of amino acids that exhibits at least 90% sequence identity to SEQ ID NO:31 or a specific binding fragment thereof.
 4. (canceled)
 5. The variant PD-L2 polypeptide of claim 1, wherein the one or more amino acid modifications are selected from F2L, I12V, I13V, H15Q, N18D, C23S, N24D, N24S, G28V, V31A, V31M, N32D, L33P, L33H, L33F, I36V, T37A, S39I, E44D, E44V, N45S, D46E, T47A, S48C, E58G, E59G, K65R, S67L, H69L, P71S, Q72H, V73A, Q74R, V75G, R76G, D77N, Q82R, I85F, I86T, V89D, W91R, or a conservative amino acid substitution thereof.
 6. The variant PD-L2 polypeptide of claim 1, wherein the one or more amino acid modifications are selected from among H15Q, N24D, E44D, V89D, Q82R/V89D, E59G/Q82R, S39I/V89D, S67L/V89D, S67L/I85F, S67L/I86T, H15Q/K65R, H15Q/Q72H/V89D, H15Q/S67L/R76G, H15Q/R76G/I85F, H15Q/T47A/Q82R, H15Q/Q82R/V89D, H15Q/C23S/I86T, H15Q/S39I/I86T, E44D/V89D/W91R, I13V/S67L/V89D, H15Q/S67L/I86T, I13V/H15Q/S67L/I86T, I13V/H15Q/E44D/V89D, I13V/S39I/E44D/Q82R/V89D, I13V/E44D/Q82R/V89D, I13V/Q72H/R76G/I86T, I13V/H15Q/R76G/I85F, H15Q/S39I/R76G/V89D, H15Q/S67L/R76G/I85F, H15Q/T47A/Q72H/R76G/I86T, H15Q/T47A/Q72H/R76G, I13V/H15Q/T47A/Q72H/R76G, H15Q/E44D/R76G/I85F, H15Q/S39I/S67L/V89D, H15Q/N32D/S67L/V89D, N32D/S67L/V89D, H15Q/S67L/Q72H/R76G/V89D, H15Q/Q72H/Q74R/R76G/I86T, G28V/Q72H/R76G/I86T, I13V/H15Q/S39I/E44D/S67L, E44D/S67L/Q72H/Q82R/V89D, H15Q/V89D, H15Q/T47A, I13V/H15Q/Q82R, I13V/H15Q/V89D, I13V/S67L/Q82R/V89D, I13V/H15Q/Q82R/V89D, H15Q/V31M/S67L/Q82R/V89D, I13V/H15Q/T47A/Q82R, I13V/H15Q/V31A/N45S/Q82R/V89D, H15Q/T47A/H69L/Q82R/V89D, I13V/H15Q/T47A/H69L/R76G/V89D, I12V/I13V/H15Q/T47A/Q82R/V89D, I13V/H15Q/R76G/D77N/Q82R/V89D, I13V/H15Q/T47A/R76G/V89D, I13V/H15Q/T47A/Q82R/V89D, I13V/H15Q/N24D/Q82R/V89D, I13V/H15Q/136V/T47A/S67L/V89D, H15Q/T47A/K65R/S67L/Q82R/V89D, H15Q/L33P/T47A/S67L/P71S/V89D, I13V/H15Q/Q72H/R76G/I86T, H15Q/T47A/S67L/Q82R/V89D, F2L/H15Q/D46E/T47A/Q72H/R76G/Q82R/V89D, I13V/H15Q/L33F/T47A/Q82R/V89D, I13V/H15Q/T47A/E58G/S67L/Q82R/V89D, H15Q/N24S/T47A/Q72H/R76G/V89D, I13V/H15Q/E44V/T47A/Q82R/V89D, H15Q/N18D/T47A/Q72H/V73A/R76G/I86T/V89D, I13V/H15Q/T37A/E44D/S48C/S67L/Q82R/V89D, H15Q/L33H/S67L/R76G/Q82R/V89D, I13V/H15Q/T47A/Q72H/R76G/I86T, H15Q/S39I/E44D/Q72H/V75G/R76G/Q82R/V89D, H15Q/T47A/S67L/R76G/Q82R/V89D, I13V/H15Q, I13V/T47A, I13V/S67L, I13V/Q72H, I13V/R76G, I13V/Q82R, I13V/I86T, I13V/V89D, H15Q/T47A, H15Q/S67L, H15Q/Q72H, H15Q/R76G, H15Q/Q82R, H15Q/I86T, H15Q/V89D, T47A/S67L, T47A/Q72H, T47A/R76G, T47A/Q82R, T47A/I86T, T47A/V89D, S67L/Q72H, S67L/R76G, S67L/Q82R, S67L/I86T, S67L/V89D, Q72H/R76G, Q72H/Q82R, Q72H/I86T, Q72H/V89D, R76G/Q82R, R76G/I86T, R76G/V89D, Q82R/I86T, Q82R/V89D, I86T/V89D, H15Q/S62L/Q82R, H15Q/S62L/V89D, H15Q/Q82R/V89D, S62L/Q82R/V89D, or I13V/H15Q/T47A/S67L/Q72H/R76G/Q82R/V89D. 7-14. (canceled)
 15. The variant PD-L2 polypeptide of any of claim 1, wherein the variant PD-L2 polypeptide specifically binds to the ectodomain of PD-1 with increased affinity compared to the binding of the unmodified PD-L2 to the ectodomain of PD-1. 16-17. (canceled)
 18. The variant PD-L2 polypeptide of claim 1 that is a soluble protein, wherein: the variant PD-L2 polypeptide lacks the PD-L2 transmembrane domain and intracellular signaling domain; and/or the variant PD-L2 polypeptide is not capable of being expressed on the surface of a cell.
 19. (canceled)
 20. The variant PD-L2 polypeptide of claim 1, wherein the variant PD-L2 polypeptide is linked to a multimerization domain.
 21. The variant PD-L2 polypeptide of claim 20, wherein the multimerization domain is an Fc domain or a variant Fc domain with reduced effector function.
 22. The variant PD-L2 polypeptide of claim 1, wherein the variant PD-L2 polypeptide is a transmembrane immunomodulatory protein comprising a transmembrane domain.
 23. The variant PD-L2 polypeptide of claim 22, further comprising a cytoplasmic signaling domain. 24-27. (canceled)
 28. An immunomodulatory protein, comprising the variant PD-L2 polypeptide of claim 1 linked to a second polypeptide comprising an immunoglobulin superfamily (IgSF) domain, or an affinity-modified IgSF domain thereof, of an IgSF family member. 29-31. (canceled)
 32. The immunomodulatory protein of claim 28, wherein the IgSF domain of the second polypeptide is an IgSF domain, or an affinity-modified IgSF domain thereof, of a ligand that binds to an inhibitory receptor. 33-34. (canceled)
 35. The immunomodulatory protein of claim 28, wherein the immunomodulatory protein further comprises a multimerization domain linked to at least one of the variant PD-L2 polypeptide or the second polypeptide. 36-40. (canceled)
 41. The immunomodulatory protein of claim 35, wherein the multimerization domain is a heterodimer. 42-49. (canceled)
 50. A conjugate, comprising a variant PD-L2 polypeptide of claim 1 linked to a targeting moiety that specifically binds to a molecule on the surface of a cell.
 51. (canceled)
 52. The conjugate of claim 50, wherein the cell is an immune cell or a tumor cell.
 53. (canceled)
 54. The conjugate of claim 50, wherein the moiety is an antibody or antigen-binding fragment.
 55. The conjugate of claim 50 that is a fusion protein.
 56. A nucleic acid molecule(s), encoding the variant PD-L2 polypeptide of claim
 1. 57. A vector, comprising the nucleic acid molecule of claim
 56. 58. (canceled)
 59. A cell, comprising the vector of claim
 57. 60. A method of producing a variant PD-L2 polypeptide or an immunomodulatory protein, comprising introducing the nucleic acid molecule of claim 56 into a host cell under conditions to express the protein in the cell.
 61. (canceled)
 62. A method of engineering a cell expressing a variant PD-L2 variant polypeptide, comprising introducing a nucleic acid molecule encoding the variant PD-L2 polypeptide of claim 1 into a host cell under conditions in which the polypeptide is expressed in the cell.
 63. An engineered cell, comprising the variant PD-L2 polypeptide of claim 1 or a nucleic acid molecule encoding a sequence of amino acids comprising the variant PD-L2 polypeptide of claim
 1. 64. The engineered cell of claim 63, wherein: the variant PD-L2 polypeptide or immunomodulatory protein is secreted or is capable of being secreted from the engineered cell; or the variant PD-L2 polypeptide is expressed on the surface of the cell.
 65. (canceled)
 66. The engineered cell of claim 63, wherein the cell is an immune cell.
 67. The engineered cell of claim 63, wherein the cell further comprises a chimeric antigen receptor (CAR) or an engineered T-cell receptor.
 68. An infectious agent, comprising a variant PD-L2 polypeptide of claim 1 or a nucleic acid molecule encoding a sequence of amino acids comprising the variant PD-L2 polypeptide of claim
 1. 69-70. (canceled)
 71. A pharmaceutical composition, comprising the variant PD-L2 polypeptide of claim
 1. 72-73. (canceled)
 74. An article of manufacture or kit comprising the pharmaceutical composition of claim
 71. 75. (canceled)
 76. A method of modulating an immune response in a subject, comprising administering the pharmaceutical composition of claim 71 to the subject.
 77. A method of modulating an immune response in a subject, comprising administering the engineered cell of claim 63 to the subject.
 78. (canceled)
 79. The method of claim 76, wherein modulating the immune response treats a disease or condition in the subject. 80.-94. (canceled)
 95. A nucleic acid molecule(s), encoding the immunomodulatory protein of claim
 28. 96. A nucleic acid molecule(s), encoding a conjugate that is a fusion protein of claim
 55. 97. A pharmaceutical composition, comprising the immunomodulatory protein of claim
 28. 98. A pharmaceutical composition, comprising the conjugate of claim
 50. 99. A pharmaceutical composition, comprising the engineered cell of claim
 63. 100. A method of modulating an immune response in a subject, comprising administering the pharmaceutical composition of claim 97 to the subject.
 101. A method of modulating an immune response in a subject, comprising administering the pharmaceutical composition of claim 98 to the subject.
 102. A method of modulating an immune response in a subject, comprising administering the pharmaceutical composition of claim 99 to the subject. 